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Phospholipase D6 (PLD6) plays pivotal roles in mitochondrial dynamics and spermatogenesis, but the cellular and subcellular localization of endogenous PLD6 in testis germ cells is poorly defined. We examined the distribution and subcellular localization of PLD6 in mouse testes using validated specific anti-PLD6 antibodies. Ectopically expressed PLD6 protein was detected in the mitochondria of PLD6-transfected cells, but endogenous PLD6 expression in mouse testes was localized to the perinuclear region of pachytene spermatocytes, and more prominently, to the round (Golgi and cap phases) and elongating spermatids (acrosomal phase); these results suggest that PLD6 is localized to the Golgi apparatus. The distribution of PLD6 in the round spermatids partially overlapped with that of the cis-Golgi marker GM130, indicating that the PLD6 expression corresponded to the GM130-positive subdomains of the Golgi apparatus. Correlative light and electron microscopy revealed that PLD6 expression in developing spermatids was localized almost exclusively to several flattened cisternae, and these structures might correspond to the medial Golgi subcompartment; neither the trans-Golgi networks nor the developing acrosomal system expressed PLD6. Further, we observed that PLD6 interacted with tesmin, a testis-specific transcript necessary for successful spermatogenesis in mouse testes. To our knowledge, these results provide the first evidence of PLD6 as a Golgi-localized protein of pachytene spermatocytes and developing spermatids and suggest that its subcompartment-specific distribution within the Golgi apparatus may be related to the specific functions of this organelle during spermatogenesis.

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) undergo extensive posttranslational modifications and remodeling, including the addition and subsequent removal of phosphoethanolamine (EtNP) from mannose 1 (Man1) and mannose 2 (Man2) of the glycan moiety. Removal of EtNP from Man1 is catalyzed by Cdc1p, an event that has previously been considered to occur in the endoplasmic reticulum (ER). We establish that Cdc1p is in fact a cis/medial Golgi membrane protein that relies on the COPI coatomer for its retention in this organelle. We also determine that Cdc1p does not cycle between the Golgi and the ER, and consistent with this finding, when expressed at endogenous levels ER-localized Cdc1p-HDEL is unable to support the growth of cdc1Δ cells. Our cdc1 temperature-sensitive alleles are defective in the transport of a prototypical GPI-AP-Gas1p to the cell surface, a finding we posit reveals a novel Golgi-localized quality control warrant. Thus, yeast cells scrutinize GPI-APs in the ER and also in the Golgi, where removal of EtNP from Man2 (via Ted1p in the ER) and from Man1 (by Cdc1p in the Golgi) functions as a quality assurance signal.

Although the general structure of the barrier between the gut and the blood is well known, many details are still missing. Here, we analyse the literature and our own data related to lipid transcytosis through adult mammalian enterocytes, and their absorption into lymph at the tissue level of the intestine. After starvation, the Golgi complex (GC) of enterocytes is in a resting state. The addition of lipids in the form of chyme leads to the initial appearance of pre-chylomicrons (ChMs) in the tubules of the smooth endoplasmic reticulum, which are attached at the basolateral plasma membrane, immediately below the 'belt' of the adhesive junctions. Then pre-ChMs move into the cisternae of the rough endoplasmic reticulum and then into the expansion of the perforated Golgi cisternae. Next, they pass through the GC, and are concentrated in the distensions of the perforated cisternae on the trans-side of the GC. The arrival of pre-ChMs at the GC leads to the transition of the GC to a state of active transport, with formation of intercisternal connections, attachment of cis-most and trans-most perforated cisternae to the medial Golgi cisternae, and disappearance of COPI vesicles. Post-Golgi carriers then deliver ChMs to the basolateral plasma membrane, fuse with it, and secret ChMs into the intercellular space between enterocytes at the level of their interdigitating contacts. Finally, ChMs are squeezed out into the interstitium through pores in the basal membrane, most likely due to the function of the actin-myosin 'cuff' around the interdigitating contacts. These pores appear to be formed by protrusions of the dendritic cells and the enterocytes per se. ChMs are absorbed from the interstitium into the lymphatic capillaries through the special oblique contacts between endothelial cells, which function as valves through the contraction-relaxation of bundles of smooth muscle cells in the interstitium. Lipid overloading of enterocytes results in accumulation of cytoplasmic lipid droplets, an increase in diameter of ChMs, inhibition of intra-Golgi transport, and fusion of ChMs in the interstitium. Here, we summarise and analyse recent findings, and discuss their functional implications.

Until now, the mechanisms of ER-to-Golgi and intra-Golgi transport remain obscure. This is especially evident for the Golgi of S. cerevisiae where different Golgi compartments are not organized in stacks. Here, using improved sample preparation protocols, we examined the 3D organization of pre-Golgi and Golgi compartments and found several new features of the structures functioning along the secretory pathway. In the cytoplasmic sheet ER, we found narrow pores that aggregated near the rims, and tubular networks tightly interconnected with sheets of several cytoplasmic ER cisternae. Within the Golgi compartments, we found disks with wide pores, disks with narrow pores, and disk-like networks with varicosities or nodules at the point of branching and thick membranes. Sometimes, these compartments contained 30 nm buds coated with a clathrin-like coat. The lumen of these Golgi compartments was more osmiophilic than the lumen of the ER. In contrast to ER elements, Golgi compartments were isolated and in the majority of cases not connected, although we observed some connections between Golgi compartments and also between Golgi disks with wide pores and the ER. Two types of free vesicles of 35-40 and 45-50 nm were found, the former being sometimes partially coated with a clathrin-like coat. Sec31, a COPII component, was found near narrow pores in the cytoplasmic sheets of the ER, over edge aggregates of narrow pores, and within the ER network. The cis-Golgi marker Rer1p was detected on disks or semi-spheres with wide pores, while the medial Golgi marker Gos1p was found on disks or semi-spheres with narrow pores. Gos1p was found to be enriched on 45-50 nm vesicles, while Rer1p was depleted. The 35-40 nm vesicles did not show either label. These findings are discussed from the point of view of mechanisms of transport.

The HA (haemagglutinin) of influenza viruses must be recruited to membrane rafts to perform its function in membrane fusion and virus budding. We previously showed using FRET that deletion of the two raft-targeting features of HA, S-acylation at the cytoplasmic tail and the hydrophobic amino acids VIL (Val-Ile-Leu) in the outer part of the TMR (transmembrane region), lead to reduced raft association. In addition, exchange of VIL, but not of the S-acylation sites severely retards transport of HA through the Golgi. In the present study, we have further characterized the ill-defined signal in the TMR. A sequence comparison suggests that the leucine residue of VIL might be part of a CCM (cholesterol consensus motif) that is known to bind cholesterol to seven-transmembrane receptors. The signal also comprises a lysine residue and a tryptophan residue on one and a tyrosine residue on another TMR helix and is conserved in group 2 HAs. Mutations in the CCM retard Golgi-localized processing of HA, such as acquisition of Endo H (endoglycosidase H)-resistant carbohydrates in the medial Golgi and proteolytic cleavage in the TGN ( trans -Golgi network). The delay in transport of HA to and from the medial Golgi varied with the mutation, suggesting that different transport steps are affected. All mutants analysed by FRET also showed reduced association with rafts at the plasma membrane. Thus the raft-targeting signal of HA encompasses not only hydrophobic, but also aromatic and positively charged, residues. We speculate that binding to cholesterol might facilitate intracellular transport of HA and association with rafts. Abbreviations: CCM, cholesterol consensus motif; Cer, Cerulean; CHO, Chinese-hamster ovary; cl., cleaved; CRAC, cholesterol-recognition/interaction amino acid consensus; DMEM, Dulbecco's modified Eagle's medium; DRM, detergent-resistant membrane; Endo H, endoglycosidase H; ER, endoplasmic reticulum; FLIM, fluorescence lifetime imaging microscopy; FPV, fowl plaque virus; gp41, glycoprotein 41; GPI, glycosylphosphatidylinositol; HA, haemagglutinin; Myr-Pal–YFP, myristoylated and palmitoylated peptide fused to YFP; TCSPC, time-correlated single-photon counting; TGN, trans-Golgi network; TMR, transmembrane region; uncl., uncleaved; wt, wild-type

In this review we discuss mucus, the viscoelastic secretion from goblet or mucous producing cells that lines the epithelial surfaces of all organs exposed to the external world. Mucus is a complex aqueous fluid that owes its viscoelastic, lubricating and hydration properties to the glycoprotein mucin combined with electrolytes, lipids and other smaller proteins. Electron microscopy of mucosal surfaces reveals a highly convoluted surface with a network of fibers and pores of varying sizes. The major structural and functional component, mucin is a complex glycoprotein coded by about 20 mucin genes which produce a protein backbone having multiple tandem repeats of Serine, Threonine (ST repeats) where oligosaccharides are covalently O-linked. The N- and C-terminals of this apoprotein contain other domains with little or no glycosylation but rich in cysteines leading to dimerization and further multimerization via SS bonds. The synthesis of this complex protein starts in the endoplasmic reticulum with the formation of the apoprotein and is further modified via glycosylation in the cis and medial Golgi and packaged into mucin granules via Ca2+ bridging of the negative charges on the oligosaccharide brush in the trans Golgi. The mucin granules fuse with the plasma membrane of the secretory cells and following activation by signaling molecules release Ca2+ and undergo a dramatic change in volume due to hydration of the highly negatively charged polymer brush leading to exocytosis from the cells and forming the mucus layer. The rheological properties of mucus and its active component mucin and its mucoadhesivity are briefly discussed in light of their importance to mucosal drug delivery.

Characterization of the structure of the N-terminal portion of yeast Sac1 in complex with the phosphatidylinositol 4-kinase effector Vps74 identifies how Sac1 is directed to dephosphorylate PtdIns4P in the Golgi., Sac1 is a phosphoinositide phosphatase of the endoplasmic reticulum and Golgi apparatus that controls organelle membrane composition principally via regulation of phosphatidylinositol 4-phosphate signaling. We present a characterization of the structure of the N-terminal portion of yeast Sac1, containing the conserved Sac1 homology domain, in complex with Vps74, a phosphatidylinositol 4-kinase effector and the orthologue of human GOLPH3. The interface involves the N-terminal subdomain of the Sac1 homology domain, within which mutations in the related Sac3/Fig4 phosphatase have been linked to Charcot–Marie–Tooth disorder CMT4J and amyotrophic lateral sclerosis. Disruption of the Sac1–Vps74 interface results in a broader distribution of phosphatidylinositol 4-phosphate within the Golgi apparatus and failure to maintain residence of a medial Golgi mannosyltransferase. The analysis prompts a revision of the membrane-docking mechanism for GOLPH3 family proteins and reveals how an effector of phosphoinositide signaling serves a dual function in signal termination.

The cisternal progression/maturation model of Golgi trafficking predicts that cis-Golgi cisternae are formed de novo on the cis-side of the Golgi. Here we describe structural and functional intermediates of the cis cisterna assembly process in high-pressure frozen algae (Scherffelia dubia, Chlamydomonas reinhardtii) and plants (Arabidopsis thaliana, Dionaea muscipula; Venus Flytrap) as determined by electron microscopy, electron tomography and immuno-electron microscopy techniques. Our findings are as follows: (1) The cis-most (C1) Golgi cisternae are generated de novo from cisterna initiators produced by the fusion of 3–5 COPII vesicles in contact with a C2 cis cisterna. (2) COPII vesicles fuel the growth of the initiators, which then merge into a coherent C1 cisterna. (3) When a C1 cisterna nucleates its first cisterna initiator it becomes a C2 cisterna. (4) C2-Cn cis cisternae grow through COPII vesicle fusion. (5) ER-resident proteins are recycled from cis cisternae to the ER via COPIa-type vesicles. (6) In S. dubia the C2 cisternae are capable of mediating the self-assembly of scale protein complexes. (7) In plants, ~90% of native α-mannosidase I localizes to medial Golgi cisternae. (8) Biochemical activation of cis cisternae appears to coincide with their conversion to medial cisternae via recycling of medial cisterna enzymes. We propose how the different cis cisterna assembly intermediates of plants and algae may actually be related to those present in the ERGIC and in the pre-cis Golgi cisterna layer in mammalian cells.

Uukuniemi virus (UUKV) is a model system for investigating the genus Phlebovirus of the Bunyaviridae . We report the UUKV glycome, revealing differential processing of the Gn and Gc virion glycoproteins. Both glycoproteins display poly- N -acetyllactosamines, consistent with virion assembly in the medial Golgi apparatus, whereas oligomannose-type glycans required for DC-SIGN-dependent cellular attachment are predominant on Gc. Local virion structure and the route of viral egress from the cell leave a functional imprint on the phleboviral glycome.

Signaling by phosphatidylinositol 4-kinases (PI4Ks) in the Golgi apparatus controls lipid homeostasis and protein-sorting pathways. Signaling is shown to be terminated on the medial cisterna by a complex of a PI4K effector, Vps74, and Sac1, the major PtdIns4P phosphatase in the cell., In the Golgi apparatus, lipid homeostasis pathways are coordinated with the biogenesis of cargo transport vesicles by phosphatidylinositol 4-kinases (PI4Ks) that produce phosphatidylinositol 4-phosphate (PtdIns4P), a signaling molecule that is recognized by downstream effector proteins. Quantitative analysis of the intra-Golgi distribution of a PtdIns4P reporter protein confirms that PtdIns4P is enriched on the trans-Golgi cisterna, but surprisingly, Vps74 (the orthologue of human GOLPH3), a PI4K effector required to maintain residence of a subset of Golgi proteins, is distributed with the opposite polarity, being most abundant on cis and medial cisternae. Vps74 binds directly to the catalytic domain of Sac1 (KD = 3.8 μM), the major PtdIns4P phosphatase in the cell, and PtdIns4P is elevated on medial Golgi cisternae in cells lacking Vps74 or Sac1, suggesting that Vps74 is a sensor of PtdIns4P level on medial Golgi cisternae that directs Sac1-mediated dephosphosphorylation of this pool of PtdIns4P. Consistent with the established role of Sac1 in the regulation of sphingolipid biosynthesis, complex sphingolipid homeostasis is perturbed in vps74Δ cells. Mutant cells lacking complex sphingolipid biosynthetic enzymes fail to properly maintain residence of a medial Golgi enzyme, and cells lacking Vps74 depend critically on complex sphingolipid biosynthesis for growth. The results establish additive roles of Vps74-mediated and sphingolipid-dependent sorting of Golgi residents.

SummaryProtein toxins such as Ricin and Pseudomonas exotoxin (PE) pose major public health challenges. Both toxins depend on host cell machinery for internalization, retrograde trafficking from endosomes to the ER, and translocation to cytosol. Although both toxins follow a similar intracellular route, it is unknown how much they rely on the same genes. Here we conducted two genome-wide RNAi screens identifying genes required for intoxication and demonstrating that requirements are strikingly different between PE and Ricin, with only 13% overlap. Yet factors required by both toxins are present from the endosomes to the ER, and, at the morphological level, the toxins colocalize in multiple structures. Interestingly, Ricin, but not PE, depends on Golgi complex integrity and colocalizes significantly with a medial Golgi marker. Our data are consistent with two intertwined pathways converging and diverging at multiple points and reveal the complexity of retrograde membrane trafficking in mammalian cells.

Glycans (i.e. oligosaccharide chains attached to cellular proteins and lipids) are crucial for nearly all aspects of life, including the development of multicellular organisms. They come in multiple forms, and much of this diversity between molecules, cells, and tissues is generated by Golgi-resident glycosidases and glycosyltransferases. However, their exact mode of functioning in glycan processing is currently unclear. Here we investigate the supramolecular organization of the N-glycosylation pathway in live cells by utilizing the bimolecular fluorescence complementation approach. We show that all four N-glycosylation enzymes tested (beta-1,2-N-acetylglucosaminyltransferase I, beta-1,2-N-acetylglucosaminyltransferase II, 1,4-galactosyltransferase I, and alpha-2,6-sialyltransferase I) form Golgi-localized homodimers. Intriguingly, the same enzymes also formed two distinct and functionally relevant heterodimers between the medial Golgi enzymes beta-1,2-N-acetylglucosaminyltransferase I and beta-1,2-N-acetylglucosaminyltransferase II and the trans-Golgi enzymes 1,4-galactosyltransferase I and alpha-2,6-sialyltransferase I. Given their strict Golgi localization and sequential order of function, the two heterodimeric complexes are probably responsible for the processing and maturation of N-glycans in live cells.

We analyzed the assembly of caveolae in CV1 cells by following the fate of newly synthesized caveolin-1 (CAV1), caveolin-2 and polymerase I and transcript release factor (PTRF)/cavin-1 biochemically and using live-cell imaging. Immediately after synthesis in the endoplasmic reticulum (ER), CAV1 assembled into 8S complexes that concentrated in ER exit sites, due to a DXE sequence in the N-terminal domain. The coat protein II (COPII) machinery allowed rapid transport to the Golgi complex. Accumulating in the medial Golgi, the caveolins lost their diffusional mobility, underwent conformational changes, associated with cholesterol, and eventually assembled into 70S complexes. Together with green fluorescent protein-glycosyl-phosphatidylinositol (GFP-GPI), the newly assembled caveolin scaffolds underwent transport to the plasma membrane in vesicular carriers distinct from those containing vesicular stomatitis virus (VSV) G-protein. After arrival, PTRF/cavin-1 was recruited to the caveolar domains over a period of 25 min or longer. PTRF/cavin-1 itself was present in 60S complexes that also formed in the absence of CAV1. Our study showed the existence of two novel large complexes containing caveolar coat components, and identified a hierarchy of events required for caveolae assembly occurring stepwise in three distinct locations--the ER, the Golgi complex and the plasma membrane.

Localization of resident Golgi proteins to earlier ( cis) or later ( trans) Golgi compartments has traditionally required quantitative immunocytochemistry and electron microscopy, which are inaccessible to many researchers. For this reason, light microscopy has often been used, initially for localization of Golgi glycotransferases and, more recently, for other Golgi proteins (e.g., Arf1, GBF1, Rab6). Quantitation of light microscopic intra-Golgi localization can be problematic. We describe here a novel quantitative light microscopic methodology using linescans crossing the Golgi ribbon. Our method determines a localization for the unknown protein in a one-dimensional coordinate system in which 0.0 corresponds to localization of a cis marker and 1.0 to localization of a trans marker. We also describe a variant of this methodology in which Golgi morphology is simplified by nocodazole-induced dispersal into ministacks, allowing a fully automated analysis. In our assay, β1,4-galactosyltransferase-YFP and Golgin97 localize similarly to trans markers, whereas p115, GBF1, and p58-YFP are similarly near other cis markers. The medial Golgi protein α1,3–1,6-mannosidase II gives an intermediate localization in this assay. These methodologies may prove useful in instances where electron microscopy is technically difficult as well as when rapid analysis of large numbers of samples is required.

We have previously shown in our laboratory that cerebrospinal fluid from ALS patients (ALS-CSF) contains putative toxic factor(s). In the present study we determined the effect of ALS-CSF on the integrity of the Golgi apparatus of spinal motor neurons in the neonatal rats. CSF was injected intrathecally into three-day-old rat pups and subsequently the ultrastructural changes in the motor neurons were studied after 48 h, 1, 2 and 3 weeks. We observed that ALS-CSF causes fragmentation of the Golgi apparatus in a considerable number of motor neurons in the spinal cord. This was further confirmed when motor neurons were stained with an antibody against a medial Golgi protein (MG160). Thus, we suggest that the putative toxin(s) present in ALS-CSF may cause impairment in the protein processing leading to motor neuron death.

Mouse GnT1IP-L, and membrane-bound GnT1IP-S (MGAT4D) expressed in cultured cells inhibit MGAT1, the N-acetylglucosaminyltransferase that initiates the synthesis of hybrid and complex N-glycans. However, it is not known where in the secretory pathway GnT1IP-L inhibits MGAT1, nor whether GnT1IP-L inhibits other N-glycan branching N-acetylglucosaminyltransferases of the medial Golgi. We show here that the luminal domain of GnT1IP-L contains its inhibitory activity. Retention of GnT1IP-L in the endoplasmic reticulum (ER) via the N-terminal region of human invariant chain p33, with or without C-terminal KDEL, markedly reduced inhibitory activity. Dynamic fluorescent resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC) assays revealed homomeric interactions for GnT1IP-L in the ER, and heteromeric interactions with MGAT1 in the Golgi. GnT1IP-L did not generate a FRET signal with MGAT2, MGAT3, MGAT4B or MGAT5 medial Golgi GlcNAc-tranferases. GnT1IP/Mgat4d transcripts are expressed predominantly in spermatocytes and spermatids in mouse, and are reduced in men with impaired spermatogenesis. DOI: http://dx.doi.org/10.7554/eLife.08916.001, eLife digest Proteins are made up of chains of amino acids that fold into three-dimensional shapes and many are assembled in a cell compartment known as the endoplasmic reticulum. From here, these new proteins move to another compartment called the Golgi, where they may be further modified before they are transported to their final destination in the cell. One way that proteins may be modified is known as glycosylation, in which sugar molecules are attached to specific amino acids. Some sugar molecules can act as labels that ensure the new proteins are transported to the correct destination in the cell. For proteins that are delivered to the surface of the cell, the sugar molecules can also play important roles in communication with other cells. A simple sugar molecule, or a complex arrangement of many sugar molecules, may be attached to an amino acid by glycosylation. An enzyme called MGAT1 controls the synthesis of sugars called complex N-glycans in the Golgi. In 2010, researchers reported that a glycoprotein called GnT1IP-L binds to MGAT1 and inhibits its activity, thereby blocking the production of complex N-glycans. GnT1IP-L was found in the endoplasmic reticulum and Golgi, but it was not clear how it inhibits MGAT1. Huang et al.—including some of the researchers from the 2010 study—have now investigated the activity of GnT1IP-L in cells grown in the laboratory using several biochemical techniques. The experiments show that GnT1IP-L only binds to MGAT1 when both proteins are in the Golgi. There are three sections (or ‘domains’) in GnT1IP-L, but Huang et al. found that only the domain that is on the inside of the Golgi is involved in this interaction. Previous work indicated that GnT1IP-L may be involved in the formation of sperm in mice. Huang et al. have now analyzed previously published data on samples of testis tissue from human patients and found that the gene that encodes GnT1IP-L is present in very low amounts in patients whose sperm do not develop properly. Huang et al.'s findings suggest that GnT1IP-L may inhibit MGAT1 to control the glycosylation of proteins in the Golgi of developing sperm. The next step is to test this hypothesis by generating mutant mice that lack GnT1IP-L, or to make GnT1P-L in other cells in which it is not normally made, to find out if this affects the production of sperm. DOI: http://dx.doi.org/10.7554/eLife.08916.002

Rab43 differentially regulates the sorting of membrane-spanning cargo in the medial Golgi. Although the trans-Golgi network is the primary site at which cargo is sorted into transport intermediates for delivery to alternative cellular compartments, cargo is also partitioned during its transit through earlier Golgi compartments., To evaluate the role of cytoplasmic domains of membrane-spanning proteins in directing trafficking through the secretory pathway, we generated fluorescently tagged VSV G tsO45 with either the native G tail (G) or a cytoplasmic tail derived from the chicken AE1-4 anion exchanger (GAE). We previously showed that these two proteins progressed through the Golgi with distinct kinetics. To investigate the basis for the differential sorting of G and GAE, we analyzed the role of several Golgi-associated small GTP-binding proteins and found that Rab43 differentially regulated their transport through the Golgi. We show that the expression of GFP-Rab43 arrested the anterograde transport of GAE in a Rab43-positive medial Golgi compartment. GFP-Rab43 expression also inhibited the acquisition of endoH-resistant sugars and the surface delivery of GAE, as well as the surface delivery of the AE1-4 anion exchanger. In contrast, GFP-Rab43 expression did not affect the glycosylation or surface delivery of G. Unexpectedly, down-regulation of endogenous Rab43 using small interfering RNA resulted in an increase in the accumulation of GAE on the cell surface while having minimal effect on the surface levels of G. Our data demonstrate that Rab43 regulates the sorting of a subset of membrane-spanning cargo as they progress through the medial Golgi.

Most inositol 1,4,5-trisphosphate receptors (IP3R) are expressed in the endoplasmic reticulum (ER), where their precise distribution underlies the spatially complex Ca2+ signals evoked by extracellular stimuli. The signals that target IP3R to the ER or, less commonly, to other membranes are unknown. We expressed yellow fluorescent protein-tagged fragments of type 1 IP3R alone or fused with a plasma membrane protein to establish the determinants of ER targeting in COS-7 cells. By using a combination of confocal imaging and glycoprotein analyses, we demonstrated that any pair of the six transmembrane domains (TMD) linked by a luminal loop retains the protein within the ER, and when attached to a plasma membrane protein (ICAM-1), prevents it from reaching the medial Golgi. TMD1 or TMD2 alone were accumulated in mitochondria, whereas TMD5 and TMD6 were retained in ER, but were unable to prevent ICAM from reaching the plasma membrane. We conclude that IP3R are targeted to the ER membrane only after synthesis of TMDs 1 and 2, and that after co-translational insertion of the remaining TMDs, redundant retention signals present in any pair of TMD retain IP3R in the ER.

In yeast primary N- and O-glycans are attached to proteins in the endoplasmic reticulum (ER), and they are elongated in the Golgi. Thus, glycan extension by Golgi enzymes has been taken as evidence for arrival of a protein in the Golgi. Two α1,6-mannosyltransferase activity-containing multiprotein complexes have been reported to recycle between the Golgi and the ER, but since resident ER proteins are not Golgi-modified, Golgi enzymes were not thought to function in the ER. Here we show that when protein exit from the ER was blocked in COPII-defective yeast mutants, the N-glycans of vacuolar carboxypeptidase Y and a set of unidentified glycoproteins were decorated with an α1,6-mannose residue, normally added in the Golgi by Och1p. Immunofluorescent staining demonstrated that Och1p accumulated in the ER under these conditions. Concomitantly, primary O-glycans of a secretory protein were extended, apparently by the medial Golgi transferase Mnt1p. Similar O-glycan extension occurred in wild-type cells when an HDEL-tagged protein was allowed to encounter glycosyltransferases in the Golgi during recycling between ER and Golgi. Golgi-specific glycosylation in the ER was reduced when Golgi-to-ER traffic was blocked, confirming that glycan extension in the ER was mainly due to recycling, rather than newly synthesized transferases.

Dicumarol (3,3'-methylenebis[4-hydroxycoumarin]) is an inhibitor of brefeldin-A-dependent ADP-ribosylation that antagonises brefeldin-A-dependent Golgi tubulation and redistribution to the endoplasmic reticulum. We have investigated whether dicumarol can directly affect the morphology of the Golgi apparatus. Here we show that dicumarol induces the breakdown of the tubular reticular networks that interconnect adjacent Golgi stacks and that contain either soluble or membrane-associated cargo proteins. This results in the formation of 65-120-nm vesicles that are sometimes invaginated. In contrast, smaller vesicles (45-65 nm in diameter, a size consistent with that of coat-protein-I-dependent vesicles) that excluded cargo proteins from their lumen are not affected by dicumarol. All other endomembranes are largely unaffected by dicumarol, including Golgi stacks, the ER, multivesicular bodies and the trans-Golgi network. In permeabilized cells, dicumarol activity depends on the function of CtBP3/BARS protein and pre-ADP-ribosylation of cytosol inhibits the breakdown of Golgi tubules by dicumarol. In functional experiments, dicumarol markedly slows down intra-Golgi traffic of VSV-G transport from the endoplasmic reticulum to the medial Golgi, and inhibits the diffusional mobility of both galactosyl transferase and VSV-G tagged with green fluorescent protein. However, it does not affect: transport from the trans-Golgi network to the cell surface; Golgi-to-endoplasmic reticulum traffic of ERGIC58; coat-protein-I-dependent Golgi vesiculation by AlF4 or ADP-ribosylation factor; or ADP-ribosylation factor and beta-coat protein binding to Golgi membranes. Thus the ADP-ribosylation inhibitor dicumarol induces the selective breakdown of the tubular components of the Golgi complex and inhibition of intra-Golgi transport. This suggests that lateral diffusion between adjacent stacks has a role in protein transport through the Golgi complex.

Previous studies from this laboratory have shown that synthesis of GT3, the precursor of c series gangliosides, occurs in proximal Golgi compartments, as has been shown for the synthesis of GM3 and GD3, the precursors of a and b series gangliosides, respectively. In this work we studied whether the synthesis of GM3, GD3, and GT3 occurs in the same or in different compartments of the proximal Golgi. For this, we examined in retina cells (a) the effect of monensin, a sodium ionophore that affects mostly the trans Golgi and the trans Golgi network function, on the metabolic labeling of glycolipids from [3H]Gal by cultured cells from 7- and 10-day chick embryos and (b) the labeling in vitro of endogenous glycolipids of Golgi membrane preparations from 7-day embryos incubated with UDP-[3H]Gal. In (a), 1 microM monensin produced a twofold accumulation of radioactive glucosylceramide and a decrease to approximately 50 and 20% of total ganglioside labeling in 7- and 10-day cells, respectively. At both ages, monensin produced a threefold accumulation of radioactive GM3 and an inhibition of > 90% of GT3, GM1, GD1a, and GT1b synthesis. GD3 synthesis was inhibited approximately 30 and 70%, respectively, in 7- and 10-day cells. In (b), > 80% of the [3H]Gal was incorporated into endogenous glucosylceramide to form radioactive lactosylceramide. About 90% of [3H]Gal-labeled lactosylceramide was converted into GM3, and most of this in turn into GD3 when unlabeled CMP-NeuAc was also present in the incubation system. Under the same conditions, however, < 5% of labeled GD3 was converted into GT3. Golgi membranes incubated with CMP-[3H]NeuAc incorporated approximately 20% of [3H]NeuAc into endogenous GT3, and this percentage was not affected by 1 microM monensin. These results indicate that synthesis of GT3 is carried out in a compartment of the proximal Golgi different from those for lactosylceramide, GM3, and GD3 synthesis. Results from the experiments with monensin point to the cis/medial Golgi as the main compartment for coupled synthesis of lactosylceramide, GM3, and GD3 and to the trans Golgi as the main compartment for synthesis of GT3.

The beta 1,6 N-acetylglucosaminyltransferase (C2GnT) has been recently mapped to the cis/medial-Golgi compartment. To analyze the Golgi-targeting determinants of C2GnT, we constructed various deletion mutants of the enzyme fused to the enhanced green fluorescent protein (EGFP) and localized these proteins by fluorescence microscopy in living cells. We found that the N-terminal peptide encompassing amino acids 1 to 32 represents the minimal Golgi-targeting signal sufficient to localize EGFP to the same compartment as the full-length C2GnT. This peptide makes up the cytoplasmic and the transmembrane domains of the enzyme and was referred to as CTd (cytoplasmic and transmembrane domains). We compared the Golgi-targeting efficiency of the C2GnT-derived CTd with its homologous domains from other glycosyltransferases, including the H-type alpha(1,2)-fucosyltransferase (FucTI), the polypeptide N-acetylgalactosaminyltransferase-I (GalNAcT-I), the alpha(1,3)-fucosyltransferase VII (FucTVII), and the alpha(2,6)-sialyltransferase (ST6Gal-I) and found that the Golgi-targeting determinants of these glycosyltransferases were also composed of their cytosolic and transmembrane domains. To investigate whether the CTd of C2GnT could serve as a cis to medial Golgi-specific signal, we tested its ability to mislocalize two late-Golgi acting glycosyltransferases FucTI and FucTVII. We show that fusing the C2GnT-derived CTd with the catalytic domain of FucTVII resulted in a complete mislocalization of the enzyme to the C2GnT compartment, with a parallel alteration of sialyl-Lewis x synthesis and P-selectin binding. The intracellular distribution and activity of FucTI, however, were not affected. Thus, CTds of either early or late-Golgi acting glycosyltransferases represent the Golgi-targeting domains of these enzymes. In addition, we show that C2GnT-derived CTd can function as a cis/medial Golgi-targeting determinant.

Tyrosine sulfation of proteins is an important post-translational modification shown to play a role in many membrane-associated or extracellular processes such as virus entry, blood clotting, antibody-mediated immune response, inflammation and egg fecundation. The sole two human enzymes that transfer sulfate moieties from 3′-phospho-adenosine-5′-phospho-sulfate onto tyrosine residues, TPST1 and TPST2, are anchored to the membranes of the trans-Golgi compartment with the catalytic domain oriented to the lumen. In contrast to the relatively well studied organization of medial Golgi enzymes, the organization of trans-Golgi transferases remains elusive. Although tyrosylprotein sulfotransferases are known to exist as homodimers in the Golgi membranes, this organization level may represent only a small piece of a puzzle that is linked to the entire picture. Here we report the formation of TPST1/TPST2 heterodimers and a novel interaction between either TPST1 or TPST2 and the α-2,6-sialyltransferase, indicating a higher organization level of tyrosylprotein sulfotransferases that may serve for substrate selectivity and/or effective organization of multiple post-translational modification of proteins.

We present evidence for two subpopulations of coatomer protein I vesicles, both containing high amounts of Golgi resident proteins but only minor amounts of anterograde cargo. Early Golgi proteins p24alpha2, beta1, delta1, and gamma3 are shown to be sorted together into vesicles that are distinct from those containing mannosidase II, a glycosidase of the medial Golgi stack, and GS28, a SNARE protein of the Golgi stack. Sorting into each vesicle population is Arf-1 and GTP hydrolysis dependent and is inhibited by aluminum and beryllium fluoride. Using synthetic peptides, we find that the cytoplasmic domain of p24beta1 can bind Arf GTPase-activating protein (GAP)1 and cause direct inhibition of ArfGAP1-mediated GTP hydrolysis on Arf-1 bound to liposomes and Golgi membranes. We propose a two-stage reaction to explain how GTP hydrolysis constitutes a prerequisite for sorting of resident proteins, yet becomes inhibited in their presence.

Cells that express multiple connexins have the capacity to form heteromeric (mixed) gap junction hemichannels. We used a dominant negative connexin construct, consisting of bacterial beta-galactosidase fused to the C terminus of connexin43 (Cx43/beta-gal), to examine connexin compatibility in NIH 3T3 cells. Cx43/beta-gal is retained in a perinuclear compartment and inhibits Cx43 transport to the cell surface. The intracellular connexin pool induced by Cx43/beta-gal colocalized with a medial Golgi apparatus marker and was readily disassembled by treatment with brefeldin A. This was unexpected, since previous studies indicated that Cx43 assembly into hexameric hemichannels occurs in the trans-Golgi network (TGN) and is sensitive to brefeldin A. Further analysis by sucrose gradient fractionation showed that Cx43 and Cx43/beta-gal were assembled into a subhexameric complex. Cx43/beta-gal also specifically interacted with Cx46, but not Cx32, consistent with the ability of Cx43/beta-gal to simultaneously inhibit multiple connexins. We confirmed that interactions between Cx43/beta-gal and Cx46 reflect the ability of Cx43 and Cx46 to form heteromeric complexes, using HeLa and alveolar epithelial cells, which express both connexins. In contrast, ROS osteoblastic cells, which differentially sort Cx43 and Cx46, did not form Cx43/Cx46 heteromers. Thus, cells have the capacity to regulate whether or not compatible connexins intermix.

A connexin construct consisting of bacterial beta-galactosidase fused to the C-terminus of connexin43 (Cx43/beta-gal) was used to examine Cx43 assembly in NIH 3T3 cells. Cx43/beta-gal is retained in a perinuclear compartment and inhibits Cx43 transport to the cell surface. The intracellular connexin pool trapped by Cx43/beta-gal was retained in a compartment that co-localized with a medial Golgi apparatus marker by immunofluorescence microscopy and that was readily disassembled by treatment with brefeldin A. Further analysis by sucrose gradient fractionation showed that Cx43 and Cx43/beta-gal were assembled into a sub-hexameric complex, and that Cx43/beta-gal expression also inhibited Cx43 assembly into hemichannels. While this is consistent with Cx43 hemichannel assembly in the trans Golgi network (TGN), these data also suggest that the dominant negative effect of Cx43/beta-gal on Cx43 trafficking may reflect a putative sub-hexameric assembly intermediate formed in the Golgi apparatus.

Membrane traffic between the endoplasmic reticulum (ER) and Golgi apparatus and through the Golgi apparatus is a highly regulated process controlled by members of the rab GTPase family. The GTP form of rab1 regulates ER to Golgi transport by interaction with the vesicle tethering factor p115 and the cis-Golgi matrix protein GM130, also part of a complex with GRASP65 important for the organization of cis-Golgi cisternae. Here, we find that a novel coiled-coil protein golgin-45 interacts with the medial-Golgi matrix protein GRASP55 and the GTP form of rab2 but not other Golgi rab proteins. Depletion of golgin-45 disrupts the Golgi apparatus and causes a block in secretory protein transport. These results demonstrate that GRASP55 and golgin-45 form a rab2 effector complex on medial-Golgi essential for normal protein transport and Golgi structure.

A central feature of cisternal progression/maturation models for anterograde transport across the Golgi stack is the requirement that the entire population of steady-state residents of this organelle be continuously transported backward to earlier cisternae to avoid loss of these residents as the membrane of the oldest (trans-most) cisterna departs the stack. For this to occur, resident proteins must be packaged into retrograde-directed transport vesicles, and to occur at the rate of anterograde transport, resident proteins must be present in vesicles at a higher concentration than in cisternal membranes. We have tested this prediction by localizing two steady-state residents of medial Golgi cisternae (mannosidase II and N-acetylglucosaminyl transferase I) at the electron microscopic level in intact cells. In both cases, these abundant cisternal constituents were strongly excluded from buds and vesicles. This result suggests that cisternal progression takes place substantially more slowly than most protein transport and therefore is unlikely to be the predominant mechanism of anterograde movement.

The plasma membrane of eukaryotic cells differs in lipid composition from most of the internal organelles, presumably reflecting differences in many of its functions. In particular, the plasma membrane is rich in sphingolipids and sterols, one property of which is to decrease the permeability and increase the thickness of lipid bilayers. In this paper, we examine the length of transmembrane domains throughout the yeast secretory pathway. Although the transmembrane domains of cis and medial Golgi residents are similar to those of endoplasmic reticulum proteins, these domains lengthen substantially beyond the medial Golgi, suggesting a thickening of the bilayer. Yeast sphingolipids have particularly long acyl chains, and Aur1p, the inositol phosphorylceramide synthase that initiates yeast sphingolipid synthesis, was found to be located in the Golgi apparatus by both immunofluorescence and membrane fractionation, with its active site apparently in the Golgi lumen. Thus, it appears that sphingolipid synthesis in yeast takes place in the Golgi, separated from glycerophospholipid synthesis in the endoplasmic reticulum. A similar separation has been found in mammalian cells, and this conservation suggests that such an arrangement of enzymes within the secretory pathway could be important for the creation of bilayers of different thickness within the cell.

To investigate the organization of Golgi glycosyltransferases and their mechanism of localization, we have compared the properties of a number of medial and late acting Golgi enzymes. The medial Golgi enzymes, N-acetylglucosaminyltransferase I and II (GnTI and GnTII) required high salt for solubilization and migrated as high molecular weight complexes on sucrose density gradients. In contrast, the late acting Golgi enzymes, beta1,4-galactosyltransferase and alpha1, 2-fucosyltransferase, were readily solubilized in low salt and migrated as monomers/dimers by sucrose density gradient centrifugation. Analysis of membrane-bound GnTI chimeras indicates that the formation of high molecular weight complexes does not require the transmembrane domain and cytoplasmic tail sequences of GnTI. Furthermore, a soluble form of GnTI, containing the stem region and catalytic domain, accumulated in the Golgi prior to secretion, in contrast to beta1,4-galactosyltransferase. Soluble GnTI, which also associated with high molecular weight complexes, was comparable with membrane-bound GnTI in its ability to glycosylate newly synthesized glycoproteins in vivo. Mutation of charged residues within the stem region of GnTI, known to be important for "kin recognition", had no effect on the efficiency of Golgi localization, the inclusion into high molecular weight complexes, nor functional activity in vivo. The differences in behavior between the medial and late acting Golgi enzymes may contribute to their differential localization and their ability to glycosylate efficiently in the correct Golgi subcompartment.

Surfactant protein B (SP-B) is essential to the function of pulmonary surfactant and to alveolar type 2 cell phenotype. Human SP-B is the 79-amino acid product of extensive post-translational processing of a 381-amino acid preproprotein. Processing involves modification of the primary translation product from 39 to 42 kDa and at least 3 subsequent proteolytic cleavages to produce the mature 8-kDa SP-B. To examine the intracellular sites of SP-B processing, we carried out immunofluorescence cytochemistry and inhibitor studies on human fetal lung in explant culture and isolated type 2 cells in monolayer culture using polyclonal antibodies to human SP-B(8) (Phe(201)-Met(279)) and specific epitopes within the N- (NFProx, Ser(145)-Leu(160); NFlank Gln(186)-Gln(200)) and C-terminal (CFlank, Gly(284)-Ser(304)) propeptides of pro-SP-B. Fluorescence immunocytochemistry using epitope-specific antisera showed colocalization of pro-SP-B with the endoplasmic reticulum resident protein BiP. The 25-kDa intermediate was partially endo H-sensitive, colocalized with the medial Golgi resident protein MG160, and shifted into the endoplasmic reticulum in the presence of brefeldin A, which interferes with anterograde transport from endoplasmic reticulum to Golgi. The 9-kDa intermediate colocalized in part with MG160 but not with Lamp-1, a transmembrane protein resident in late endosomes and lamellar bodies. Brefeldin A induced a loss of colocalization between MG160 and NFlank, shifting NFlank immunostaining to a juxtanuclear tubular array. In pulse-chase studies, brefeldin A blocked all processing of 42-kDa pro-SP-B whereas similar studies using monensin blocked the final N-terminal processing event of 9 to 8 kDa SP-B. We conclude that: 1) the first enzymatic cleavage of pro-SP-B to the 25-kDa intermediate is in the brefeldin A-sensitive, medial Golgi; 2) cleavage of the 25-kDa intermediate to a 9-kDa form is a trans-Golgi event that is slowed but not blocked by monensin; 3) the final cleavage of 9 to 8 kDa SP-B is a monensin-sensitive, post-Golgi event occurring prior to transfer of SP-B to lamellar bodies.

VIP36, an integral membrane protein previously isolated from epithelial MDCK cells, is an intracellular lectin of the secretory pathway. Overexpressed VIP36 had been localised to the Golgi complex, plasma membrane and endocytic structures suggesting post-Golgi trafficking of this molecule (Fiedler et al., 1994). Here we provide evidence that endogenous VIP36 is localised to the Golgi apparatus and the early secretory pathway of MDCK and Vero cells and propose that retention is easily saturated. High resolution confocal microscopy shows partial overlap of VIP36 with Golgi marker proteins. Punctate cytoplasmic structures colocalise with coatomer and ERGIC-53, labeling ER-Golgi intermediate membrane structures. Cycling of VIP36 is suggested by colocalisation with anterograde cargo trapped in pre-Golgi structures and modification of its N-linked carbohydrate by glycosylation enzymes of medial Golgi cisternae. Furthermore, after brefeldin A treatment VIP36 is segregated from resident Golgi proteins and codistributes with ER-Golgi recycling proteins.

To gain an insight in the routing, processing and export of rat prolactin, rat pituitary cells were cultured in serum-free medium in the presence of cycloheximide, carbonyl cyanide m-chlorophenylhydrazone, Brefeldin A and monensin. The potential influence of these perturbants, whose well documented effects are the altering of protein synthesis and transport, was studied on rat prolactin molecular size isoforms appearing in cellular extracts and in culture medium. The outcome of the culture experiments as recorded in vertical SDS-PAGE, thiol gradient electrophoresis and sequential SDS-PAGE followed by prolactin specific immunoblotting and densitometry, was as follows: (1) at the cellular level we were able to characterize a novel 36 kDa protein as a disulphide-bridged oligomeric precursor prolactin, which is presumably rapidly transformed in the cis/medial Golgi; to designate monomeric rat prolactin as an early Golgi protein and t o advance evidence that the main processing of the glycosylated rat prolactin is a cis/medial Golgi event; (2) in release none of the perturbants disturbed the relative distribution of monomeric and glycosylated rat prolactin, the main molecular size isoforms currently secreted by untreated pituitary cells, or induced the appearance of transformed molecular size isoforms; (3) the secretion mode indicates that rat prolactin is released via the regulated pathway in the presence of the perturbants used.

Since its first description over a century ago, the Golgi apparatus has received a great deal of interest. Despite considerable advances in identifying key components during the past decades, the molecular mechanisms underlying the maintenance of the structural organization and functional integrity of this dynamic organelle are not yet really understood. Our laboratory recently proposed that Naa60 might be a player in these processes since we found Naa60-depleted cells to suffer structural abnormalities of the Golgi apparatus [1]. Naa60 (Nα-acetyltransferase 60) belongs to a family of enzymes, the N-terminal acetyltransferases (NATs) that catalyze acetylation of the Nα-group that is present on all protein N-termini (Nt-acetylation) [2]. For several proteins, an acetylated N-terminus is essential either for its half-life, subcellular targeting or interaction properties [3, 4]. At the cellular level, Nt-acetylation has emerged as a crucial protein modification since NAT-lacking cells suffer severe malfunctions [4]. Also, the physiological significance of Nt-acetylation has become apparent through recently discovered diseases caused by genetic defects or deficiencies in NAT enzymes [4]. But still we know little about how the NATs perform these tasks at the molecular level. Our recent study [1] demonstrated Naa60 to be an original among the NATs. Firstly, Naa60 (also called NatF) drastically differed from the other known human NATs in terms of its subcellular localization. Naa60 had an organellar localization, mainly to the Golgi, whereas the other NATs had cytosolic and cytosolic/nuclear localization patterns. Secondly, we found Naa60 to preferentially Nt-acetylate transmembrane proteins. This is a completely new concept in NAT substrate specificity, which is generally defined by the few first N-terminal amino acids, not by the protein's residency. Of notice, we found Naa60 to act on protein N-termini facing the cytosolic side of various intracellular membranes and the topological position of Naa60 matched accordingly [1]. Thirdly, the abovementioned features of Naa60 indirectly suggest that it might diverge from the established dogma of co-translational enzymatic activity for the NATs. Based on these characterizations of Naa60 we entitled it the first organellar N-terminal acetyltransferase. Whereas more than 80% of all soluble human proteins have previously been estimated to be Nt-acetylated [5], transmembrane proteins have been a thus far poorly characterized part of the Nt-acetylome. Interestingly, in addition to identifying particular transmembrane proteins as substrates of Naa60, we further showed that Nt-acetylation turns out to be a frequent modification also among transmembrane proteins [1]. Thus this work emphasizes the widespread nature of this somewhat under-acknowledged protein modification by demonstrating its applicability also to the transmembrane and organellar proteins. In addition to Naa60's presence at the Golgi, our study [1] also indicated an important role of Naa60 related to the structural integrity of this organelle. Knockdown of Naa60 caused the Golgi to fracture into smaller, more dispersed substructures, suggesting these cells to lack the structural organization that is referred to as the Golgi ribbon. This higher-ordered structural entity is made up of multiple cisternal stacks that are connected laterally into a ribbon-like structure near the perinuclear region. Additional confocal fluorescence microscopy analysis of Naa60-deficient cells revealed that cis and medial Golgi marker proteins remained in close proximity, hence suggesting cisternal stacking to be preserved, but the ribbon to be unlinked and dispersed into mini-stacks. The morphological organization of the Golgi apparatus varies among different cell types and species, with the ribbon-shaped structure typically appearing in vertebrates, whereas lower eukaryotes typically have a less complex organization of their Golgi [6]. Interestingly, Naa60 also has a similar evolutionary profile, as it is absent from yeast and unicellular eukaryotes. Conceivably, Naa60 could have co-evolved with the increased complexity of the Golgi. The functional advantage of the linked ribbon structure is still uncertain. It is not required for general membrane trafficking and secretion, and a potential role in glycosylation is unclear, however it is essential in polarization of the secretory pathway [6]. In terms of understanding the function of Naa60 at the Golgi, it is interesting to consider which proteins might potentially require the Nt-acetyl group that is provided by Naa60. A large number of tethering factors and linker proteins contribute directly to the stacking of Golgi cisternae and formation of the Golgi ribbon [7]. Also, the dynamic nature of the Golgi makes the balance of membrane trafficking, and thus proteins of the transport machinery, essential in the maintenance of Golgi structural organization [7]. In addition, Golgi positioning and organization depend on microtubules and motor proteins [7]. How the presence of Naa60 is essential for the property of the Golgi to form or maintain the ribbon structure is yet unanswered. However, based on available data, it seems reasonable to infer that it may involve the Naa60-mediated Nt-acetylation of membrane proteins with key functions in this process. The proposed model of Naa60 functioning is shown in Figure ​Figure11. Figure 1 A model of Naa60's function in Golgi ribbon maintenance

Members of the Bunyaviridae family acquire an envelope by budding through the lipid bilayer of the Golgi complex. The budding compartment is thought to be determined by the accumulation of the two heterodimeric membrane glycoproteins G1 and G2 in the Golgi. We recently mapped the retention signal for Golgi localization in one Bunyaviridae member (Uukuniemi virus) to the cytoplasmic tail of G1. We now show that a myc-tagged 81-residue G1 tail peptide expressed in BHK21 cells is efficiently targeted to the Golgi complex and retained there during a 3-h chase. Green-fluorescence protein tagged at either end with this peptide or with a C-terminally truncated 60-residue G1 tail peptide was also efficiently targeted to the Golgi. The 81-residue peptide colocalized with mannosidase II (a medial Golgi marker) and partially with p58 (an intermediate compartment marker) and TGN38 (a trans -Golgi marker). In addition, the 81-residue tail peptide induced the formation of brefeldin A-resistant vacuoles that did not costain with markers for other membrane compartments. Removal of the first 10 N-terminal residues had no effect on the Golgi localization but abolished the vacuolar staining. The shortest peptide still able to become targeted to the Golgi encompassed residues 10 to 40. Subcellular fractionation showed that the 81-residue tail peptide was associated with microsomal membranes. Removal of the two palmitylation sites from the tail peptide did not affect Golgi localization and had only a minor effect on the association with microsomal membranes. Taken together, the results provide strong evidence that Golgi retention of the heterodimeric G1-G2 spike protein complex of Uukuniemi virus is mediated by a short region in the cytoplasmic tail of the G1 glycoprotein.

Intracellular protein traffic involves a tightly regulated series of events in which a membrane-bounded vesicles bud from one compartment and are specifically targeted to the next compartment, where they dock and fuse. A cell-free system that reconstitutes vesicle trafficking between the cis and medial Golgi cisternae has been used previously to identify several proteins involved in vesicular transport (N-ethylmaleimide-sensitive fusion protein, soluble N-ethylmaleimide-sensitive fusion protein attachment proteins, p115, and p16); however, these factors are insufficient to drive the transport reaction. We have used a modified version of this in vitro intra-Golgi transport assay to guide purification of a new transport-stimulating activity. The active component is a 13 S hetero-oligomeric complex consisting of at least five polypeptides (approximately 110, 109, 90, 82, and 71 kDa), which we term Golgi transport complex (GTC). Hydrodynamic properties suggest that GTC is approximately 800 kDa and nonglobular. We obtained peptide sequence information from the 90-kDa subunit (GTC-90) that allowed us to identify a number of GTC-90 cDNAs. Comparison of these cDNAs with one another and with the genomic sequence suggests that the GTC-90 mRNA is alternatively spliced. Anti-GTC-90 antibodies inhibit the in vitro Golgi transport assay, confirming the functionality of the purified complex. Subcellular fractionation indicates that GTC-90 exists in both membrane and cytosolic pools, with the cytosolic pool associated exclusively with the GTC complex. The membrane-associated pool of GTC-90 is localized to the Golgi apparatus.

Ypt6p, the yeast homologue of human RAB6, is required for protein trafficking at elevated temperatures. Biochemical data provide evidence that Ypt6p plays a role in an early step(s) of the secretory pathway: from ER to Golgi, or from cis to medial Golgi, or both. Here we show that overexpression of YPT1 suppresses the growth and secretion defects of a ypt6 temperature-sensitive (ts) strain. SLY1-20, encoding a dominant mutant allele that suppresses the lethal effect of YPT1, also suppresses the growth defect of a ypt6 ts strain. Conversely, SSD1, isolated as a suppressor of ypt6 ts, can suppress the growth defect of a ypt1 ts allele. These data suggest that Ypt6p has some redundant function with Ypt1p. However, overexpression of Ypt6p is toxic to a ypt1 ts strain, although it does not affect the growth of wild-type cells, suggesting that Ypt6p may sequester proteins shared with Ypt1p. This genetic evidence confirms the conclusion that Ypt6p is involved in an early step of the secretory pathway. © 1998 John Wiley & Sons, Ltd.

Small GTP-binding proteins of the Rab family play important roles at defined steps of vesicular transport in protein secretion and the endocytosis pathway. In mammals, more than 30 proteins belonging to the Rab family have been reported to date. We report here the molecular cloning and characterization of a novel Rab protein, Rab33B. The amino acid sequence of Rab33B shows 55.3% identity to the Rab33A protein (previously called S10), and these two proteins share unique amino acid sequences at the effector domain. The genomic organization of rab33B was the same as rab33A: it consists of two exons. Thus, these two proteins make a subclass within the Rab family. Northern blot analysis showed that rab33B is expressed ubiquitously in mouse tissues, in contrast to rab33A whose expression is restricted to the brain and the immune system. A 26 kDa protein was detected by western blotting using a Rab33B-specific monoclonal antibody. Using immunofluorescence studies, Rab33B was shown to co-localize with (alpha)-mannosidase II, a Golgi-specific marker. Immunoelectron microscopy analysis further defined the localization of Rab33B to the medial Golgi cisternae. These results suggest Rab33B plays a role in intra-Golgi transport.

MG160, a type I cysteine-rich membrane sialoglycoprotein residing in the medial cisternae of the rat Golgi apparatus, is highly homologous to CFR, a fibroblast growth factor receptor, and ESL-1, an E-selectin ligand located at the cell surface of mouse myeloid cells and recently detected in the Golgi apparatus as well. The mechanism for the transport of MG160 from the Golgi apparatus to the cell surface is unknown. In this study we found that differential processing of the carboxy-terminal cytoplasmic domain (CD), consisting of amino acids Arg1159 Ile Thr Lys Arg Val Thr Arg Glu Leu Lys Asp Arg1171, resulted in the partial transport of the protein to the plasma membrane and filopodia. In Chinese hamster ovary cells (CHO), stably transfected with the entire cDNA encoding MG160, the protein was localized in the Golgi apparatus. However, when the terminal Arg1171 or up to nine distal amino acids were deleted, the protein was distributed to the plasma membrane and filopodia as well as the Golgi apparatus. This report shows that the CD of an endogenous type I Golgi protein is important for its efficient retention and identifies a unique residue preference in this process. Cleavage within the CD of MG160 may constitute a regulatory mechanism for the partial export of the protein from the Golgi apparatus to the plasma membrane and filopodia.

Biosynthesis of plasma membrane sphingolipids involves the coordinate action of enzymes localized to individual compartments of the biosynthetic secretory pathway of proteins. These stations include the endoplasmic reticulum and the Golgi apparatus. Although a precise localization of all the enzymes that synthesize glycosphingolipids has not been achieved to date, it is assumed that the sequence of events in glycosphingolipid biosynthesis resembles that in glycoprotein biosynthesis, i.e. that early reactions occur in early stations (endoplasmic reticulum and cis/medial Golgi) of the pathway, and late reactions occur in late stations (trans Golgi/trans Golgi network). Using truncated analogues of ceramide and glucosylceramide that allow measurement of enzyme activities in intact membrane fractions, we have reinvestigated the localization of individual enzymes involved in glycosphingolipid biosynthesis and for the first time studied the localization of lactosylceramide synthase after partial separation of Golgi membranes as previously described (Trinchera, M., and Ghidoni, R. (1989) J. Biol. Chem. 264, 15766–15769). Here, we show that the reactions involved in higher glycosphingolipid biosynthesis, including lactosylceramide synthesis, all reside in the lumen of the late Golgi compartments from rat liver.

The role of microtubules in the brefeldin A (BFA)-associated relocation of major histocompatibility complex (MHC) class II alphabeta chains (alphabeta) and the invariant chain (Ii) was characterized in Raji cells by the use of nocodazole (ND). BFA blocked the transport of alphabeta Ii proteins through the Golgi and redistributed them to the endoplasmic reticulum (ER) along with Golgi-resident enzymes. The result of the colocalization of processing enzymes and newly synthesized proteins was a downshift of alphabeta Ii molecular weight (MW) of 2 kDa, and their resistance to endoglycosidase H (endo H) after 6 hr of chase. ND by itself had no effect on the processing and transport of alphabeta to the cell surface. The addition of ND to BFA-treated cells downshifted alphabeta Ii by 4 kDa. Additionally, alphabeta Ii proteins remained sensitive to neuraminidase after 16 hr of chase. In vitro alpha-mannosidase treatment of immunoprecipitated alphabeta Ii generated a similar 4-kDa downshift of MW. Either 1-deoxymannojirimycin (DJN) or swainsonine (SWN) blocked the MW downshift caused by BFA + ND treatment. These observations indicated that in Raji cells, most of the BFA-associated relocations of cis-, medial Golgi proteins, and the addition of sialic acid from the trans-Golgi were microtubule-independent. The retrograde transport of the medial Golgi enzyme N-acetylglucosamine transferase, however, required microtubular function. Microtubule disrupters could affect BFA treatment of viral infections by further disrupting viral protein processing.

We have compared the effect of mislocalization of a Golgi glycosyltransferase in heterologous and homologous cell systems on the distribution of other Golgi-associated proteins. Mycspacer-human N-acetylglucosaminyltransferase I (NAGT-I), an N-terminally epitope-tagged NAGT-I, in which the first added negatively charged amino acid is in position 13, localizes to the endoplasmic reticulum (ER) by immunofluorescence when expressed in monkey (Vero) or human (HeLa) cells. When myc-spacer-human NAGT-I was expressed in Vero cells, the distribution of the Golgi-associated coat protein, beta-COP, was concentrated juxtanuclearly and undisturbed relative to control. When myc-spacer-human NAGT-I was expressed in HeLa cells, however, both endogenous beta-COP and GalT were no longer concentrated in a juxtanuclear manner but were rather cytoplasmically distributed as was the myc-tagged human NAGT-I. Based on these observation, we suggest that extensive interactions between proteins that normally show overlapping distributions between the medial Golgi stack and trans Golgi/TGN are possible. Moreover, we suggest that small differences in sequence may play a large role in potentiating interactions of Golgi complex proteins.

The small GTP-binding protein Rab6, a member of the Ras superfamily, is localized on the membranes of the Golgi apparatus and the trans Golgi network. Recent studies revealed that the Rab6 protein might be involved in the transit of proteins through the Golgi complex. In this report we demonstrate the essential function of the Rab6 protein in a distinct step of reconstituted Golgi transport. Polyclonal antibodies and Fab fragments directed against the C-terminal part of the Rab6 protein inhibit transport between the cis and the medial Golgi cisternae. Inhibition also occurred when a trans-dominant mutant form of the Rab6 protein (N126I) was added to the reconstituted transport. Furthermore, Rab6 antibodies inhibit uncoupled fusion of Golgi membranes. From these data we conclude that Rab6 is involved in a process related to membrane fusion at the cisternal membranes of the Golgi apparatus and therefore is needed for the consumption of Golgi-derived vesicles by their target membranes.

High level expression of the M2 ion channel protein of influenza virus inhibits the rate of intracellular transport of the influenza virus hemagglutinin (HA) and that of other integral membrane glycoproteins. HA coexpressed with M2 is properly folded, is not associated with GRP78-BiP, and trimerizes with the same kinetics as when HA is expressed alone. Analysis of the rate of transport of HA from the ER to the cis and medial golgi compartments and the TGN indicated that transport through the Golgi apparatus is delayed. Uncleaved HA0 was not expressed at the cell surface, and accumulation HA at the plasma membrane was reduced to 75-80% of control cells. The delay in intracellular transport of HA on coexpression of M2 was not observed in the presence of the M2-specific ion channel blocker, amantadine, indicating that the Golgi transport delay is due to the M2 protein ion channel activity equilibrating pH between the Golgi lumen and the cytoplasm, and not due to saturation of the intracellular transport machinery. The Na+/H+ ionophore, monensin, which also equilibrates pH between the Golgi lumen and the cytoplasm, caused a similar inhibition of intracellular transport as M2 protein expression did for HA and other integral membrane glycoproteins. EM data showed a dilation of Golgi cisternae in cells expressing the M2 ion channel protein. Taken together, the data suggest a similarity of effects of M2 ion channel activity and monensin on intracellular transport through the Golgi apparatus.

The yeast Kre2p/Mnt1p alpha 1,2-mannosyltransferase is a type II membrane protein with a short cytoplasmic amino terminus, a membrane-spanning region, and a large catalytic luminal domain containing one N-glycosylation site. Anti-Kre2p/Mnt1p antibodies identify a 60-kD integral membrane protein that is progressively N-glycosylated in an MNN1-dependent manner. Kre2p/Mnt1p is localized in a Golgi compartment that overlaps with that containing the medial-Golgi mannosyltransferase Mnn1p, and distinct from that including the late Golgi protein Kex1p. To determine which regions of Kre2p/Mnt1p are required for Golgi localization, Kre2p/Mnt1p mutant proteins were assembled by substitution of Kre2p domains with equivalent sequences from the vacuolar proteins DPAP B and Pho8p. Chimeric proteins were tested for correct topology, in vitro and in vivo activity, and were localized intracellularly by indirect immunofluorescence. The results demonstrate that the NH2-terminal cytoplasmic domain is necessary for correct Kre2p Golgi localization whereas, the membrane-spanning and stem domains are dispensable. However, in a test of targeting sufficiency, the presence of the entire Kre2p cytoplasmic tail, plus the transmembrane domain and a 36-amino acid residue luminal stem region was required to localize a Pho8p reporter protein to the yeast Golgi.

The single cell-layer of aleurone cells exhibits structural evidence of high levels of Golgi apparatus mediated secretory activity [1]. We have used an antiserum directed against β-1,2-xyloglycan side chains of proteins as a probe of protein trafficking and Golgi activity. Although the aleurone contains many distinct xyloglycoproteins the primary xyloglycoprotein accumulated in the aleurone is a 42-kDa polypeptide. The 42-kDa xyloglycoprotein is accumulated in protein storage vacuoles within the aleurone cells. Comparison of anti-β-xyloglycoprotein immunoblots of the other embryonic tissues, cotyledon and axis, indicated that while numerous xyloglycoproteins appeared to be present in these tissues the 42-kDa xyloglycoprotein was absent. The identity of the protein storage vacuoles was confirmed by the presence of α-TIP (α-tonoplast integral protein) in the limiting membrane of the vacuoles. The aleurone also accumulates a variety of embryo-specific proteins including conglycinin, soybean agglutinin, and the oil body protein 24-kDa oleosin. The xyloglycoproteins appear to traffic through the Golgi apparatus. The anti-β-xyloglycoprotein serum labeled xyloglycoproteins in the medial-trans Golgi cisternae suggesting that the xylosylation of N -linked glycan was a medial-late processing event initiated in the medial Golgi cisternae. We interpret our results to indicate soybean aleurone cells accumulate a specific abundant xyloglycoprotein of unknown function in the cell's protein storage vacuole that appears to be unique to this single-cell layer of embryonic cells.

The distribution of beta 1,2 N-acetylglucosaminyltransferase I (NAGT I), alpha 1,3-1,6 mannosidase II (Mann II), beta 1,4 galactosyltransferase (GalT), alpha 2,6 sialyltransferase (SialylT) was determined by immuno-labelling of cryo-sections from HeLa cell lines. Antibody labelling in the HeLa cell line was made possible by stable expression of epitope-tagged forms of these proteins or forms from species to which specific antibodies were available. NAGT I and Mann II had the same distribution occupying the medial and trans cisternae of the stack. GalT and SialylT also had the same distribution but they occupied the trans cisterna and the trans-Golgi network (TGN). These results generalise our earlier observations on the overlapping distribution of Golgi enzymes and show that each of the trans compartments of the Golgi apparatus in HeLa cells contains unique mixtures of those Golgi enzymes involved in the construction of complex, N-linked oligosaccharides.

Rab GTPases are master regulators of membrane trafficking events and template the directionality of protein transport through the secretory and endocytic pathways. Certain Rabs recruit the guanine nucleotide exchange factor (GEF) that activates a subsequent acting Rab protein in a given pathway; this process has been termed a Rab cascade. We show here that the medial Golgi-localized Rab33B GTPase has the potential to link functionally to the late Golgi, Rab6 GTPase, by its capacity for association with Ric1 and Rgp1 proteins. In yeast, Ric1p and Rgp1p form a complex that catalyzes guanine nucleotide exchange by Ypt6p, the Rab6 homolog. Human Ric1 and Rgp1 both bind Rab6A with preference for the GDP-bound conformation, characteristic of a GEF. Nevertheless, both Ric1 and Rgp1 proteins are needed to catalyze nucleotide exchange on Rab6A protein. Ric1 and Rgp1 form a complex, but unlike their yeast counterparts, most of the subunits are not associated, and most of the proteins are cytosolic. Loss of Ric1 or Rgp1 leads to destabilization of Rab6, concomitant with a block in Rab6-dependent retrograde transport of mannose 6-phosphate receptors to the Golgi. The C terminus of Ric1 protein contains a distinct binding site for Rab33B-GTP, supporting the existence of a Rab cascade between the medial and trans Golgi. This study thus identifies a GEF for Rab6A in human cells.

Posttranslational modification of yeast glycoproteins with alpha 1,3-linked mannose is initiated within a Golgi compartment analogous to the medial Golgi cisternae of higher eukaryotes. We have characterized the synthesis, posttranslational modification, and localization of the yeast alpha 1,3 mannosyltransferase (Mnn1p) using antibodies prepared against a segment of this protein expressed in bacteria. Mnn1p is initially synthesized as a 98.5-kD, type II integral membrane glycoprotein that is modified with both N- and O-linked oligosaccharides. It is subject to a slow, incremental increase in molecular mass that is dependent upon protein transport to the Golgi complex. Self-modification of Mnn1p with alpha 1,3 mannose epitopes, primarily on O-linked oligosaccharides, is at least partly responsible for the incremental increase in molecular mass. Mnn1p is a resident protein of the Golgi complex and colocalizes with guanosine diphosphatase to at least two physically distinct Golgi compartments by sucrose gradient fractionation, one of which may be a late Golgi compartment that also contains the Kex2 endopeptidase. Surprisingly, we found that a significant fraction of Mnn1p is mislocalized to the plasma membrane in a clathrin heavy chain temperature sensitive mutant while guanosine diphosphatase remains intracellular. A mutant Mnn1p that lacks the NH2-terminal cytoplasmic tail is properly localized to the Golgi complex, indicating that clathrin does not mediate Mnnlp Golgi retention by a direct interaction with the Mnn1p cytoplasmic tail. These results indicate that clathrin plays a broader role in the localization of Golgi proteins than anticipated.

Immunolocalization of two highly acidic polysaccharides (PS-1 and PS-2) in a calcifying algaPleurochrysis carterae is described throughout the mineralization process, from before crystal nucleation through the cessation of crystal growth. This unicellular coccolithophorid alga is a useful model for mineralization because it produces calcified scales known as coccoliths in homogeneous cell culture. PS-1 and PS-2 were localized in the crystal coats of mature coccoliths and in electron dense Golgi particles. The polyanions are synthesized in medial Golgi cisternae and co-aggregate with calcium ions into discrete 25 nm particles. Particle-laden vesicles bud from cisternal margins and fuse with a coccolith-forming saccule containing an organic oval-shaped scale which forms the base of the future coccolith. The particles are localized on the base before the onset of mineral deposition and are present in the coccolith saccule throughout the period of crystal (CaCO3) nucleation and growth. During the final phase of coccolith formation, the particles disappear, and the mature crystals acquire an amorphous coat containing PS-1 and PS-2 polysaccharides which remain with the mineral phase after the coccoliths are extruded from the cell. Postulated mechanisms of polyanion-mediated mineralization are reviewed and their relevance to the calcification of coccoliths is addressed.