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Spectrin (βIΣ∗) and ankyrin (Ank G119 ) associate with Golgi membranes and the dynactin complex, but their role in vesicle trafficking remains uncertain. We find that the actin-binding domain and membrane-association domain 1 (MAD1) of βI spectrin together form a constitutive Golgi targeting signal in transfected MDCK cells. Expression of this signal in transfected cells disrupts the endogenous Golgi spectrin skeleton and blocks transport of α- and β-Na,K-ATPase and vesicular stomatitis virus-G protein from the endoplasmic reticulum (ER) but does not disrupt the formation of Golgi stacks, the distribution of β-COP, or the transport and surface display of E-cadherin. The Golgi spectrin skeleton is thus required for the transport of a subset of membrane proteins from the ER to the Golgi. We postulate that together with polyfunctional adapter proteins such as Ank G119 , Golgi spectrin forms a docking complex that acts prior to the cis-Golgi, presumably with vesicular–tubular clusters (VTCs or ERGIC), to sequester specific membrane proteins into vesicles transiting between the ER and Golgi, and subsequently (probably involving other isoforms of spectrin and ankyrin) to mediate cargo transport within the Golgi and to other membrane compartments. We hypothesize that this vesicular spectrin–ankyrin adapter-protein trafficking (or tethering) system (SAATS) mediates the capture and transport of many membrane proteins and acts in conjunction with vesicle-targeting molecules to effect the efficient transport of cargo proteins.

When alpha(1,2)fucosyltransferase cDNA is expressed in cells that normally express large amounts of the terminal carbohydrate Galalpha(1,3)Gal, and therefore the alpha(1,3)galactosyltransferase (GT), the Galalpha(1,3)Gal almost disappears, indicating that the presence of the alpha(1,2)fucosyltransferase (HT) gene/enzyme alters the synthesis of Galalpha(1,3)Gal. A possible mechanism to account for these findings is enzyme location within the Golgi apparatus. We examined the effect of Golgi localization by exchanging the cytoplasmic tails of HT and GT; if Golgi targeting signals are contained within the cytoplasmic tail sequences of these enzymes then a "tail switch" would permit GT first access to the substrate and thereby reverse the observed dominance of HT. Two chimeric glycosyltransferase proteins were constructed and compared with the normal glycosyltransferases after transfection into COS cells. The chimeric enzymes showed Km values and cell surface carbohydrate expression comparable with normal glycosyltransferases. Co-expression of the two chimeric glycosyltransferases resulted in cell surface expression of Galalpha(1,3)Gal, and virtually no HT product was expressed. Thus the cytoplasmic tail of HT determines the temporal order of action, and therefore dominance, of these two enzymes.

The mechanism by which Golgi membrane proteins are retained within the Golgi complex in the midst of a continuous flow of protein and lipid is not yet understood. The diffusional mobilities of mammalian Golgi membrane proteins fused with green fluorescent protein from Aequorea victoria were measured in living HeLa cells with the fluorescence photobleaching recovery technique. The diffusion coefficients ranged from 3 × 10 −9 square centimeters per second to 5 × 10 −9 square centimeters per second, with greater than 90 percent of the chimeric proteins mobile. Extensive lateral diffusion of the chimeric proteins occurred between Golgi stacks. Thus, the chimeras diffuse rapidly and freely in Golgi membranes, which suggests that Golgi targeting and retention of these molecules does not depend on protein immobilization.

The mouse 8.5 mRNA encodes a 171-residue novel protein which displays a highly significant similarity with the product of the previously characterized neuronal p1A75 cDNA (Sutcliffe, J. G., Milner, R. J., Shinnick, T. M., and Bloom, F. E.(1983) Cell 33, 671-682). Northern blot and in situ hybridization experiments indicated that the 8.5 mRNA is specifically expressed in neural and neuroendocrine tissues. An affinity-purified antibody directed against the recombinant 8.5 protein demonstrated the existence of the 19-kDa natural protein in brain and evidenced its prominent juxtanuclear Golgi-like localization in cultured neurons. Ultrastructural analysis of the same preparation revealed a specific labeling of all the Golgi saccules and of some vesicles in the Golgi zone. In transfected COS cells, the exogenous protein was also detected in the Golgi area, indicating, therefore, the presence of a Golgi targeting signal in its primary sequence.

Rubella virus (RV) envelope glycoproteins, E2 and E1, form a heterodimeric complex that is targeted to medial/trans-Golgi cisternae. To identify the Golgi targeting signal(s) for the E2/E1 spike complex, we constructed chimeric proteins consisting of domains from RV glycoproteins and vesicular stomatitis virus (VSV) G protein. The location of the chimeric proteins in stably transfected Chinese hamster ovary cells was determined by immunofluorescence, immunoelectron microscopy, and by the extent of processing of their N-linked glycans. A trans-dominant Golgi retention signal was identified within the C-terminal region of E2. When the transmembrane (TM) and cytoplasmic (CT) domains of VSV G were replaced with those of RV E2, the hybrid protein (G-E2TMCT+) was retained in the Golgi. Transport of G-E2TMCT+ to the Golgi was rapid (t1/2 = 10-20 min). The G-E2TMCT+ protein was determined to be distal to or within the medial Golgi based on acquisition of endo H resistance but proximal to the trans-Golgi network since it lacked sialic acid. Deletion analysis revealed that only the TM domain of E2 was required for Golgi targeting. Although the cytoplasmic domain of E2 was not necessary for Golgi retention, it was required for efficient transport of VSV G-RV chimeras out of the endoplasmic reticulum. When assayed in sucrose velocity sedimentations gradients, the Golgi-retained G-E2TMCT+ protein behaved as a dimer. Unlike virtually all other Golgi targeting signals, the E2 TM domain does not contain any polar amino acids. The TM and CT domains of E1 were not required for targeting of E2 and E1 to the Golgi indicating that a heterodimer of two integral membrane proteins can be retained in the Golgi by a single retention signal.

To investigate several key aspects of phosphatidylinositol transfer protein (PI-TP) function in eukaryotic cells, rat PI-TP was expressed in yeast strains carrying lesions in SEC14, the structural gene for yeast PI-TP (SEC14p), whose activity is essential for Golgi secretory function in vivo. Rat PI-TP expression effected a specific complementation of sec14ts growth and secretory defects. Complementation of sec14 mutations was not absolute as rat PI-TP expression failed to rescue sec14 null mutations. This partial complementation of sec14 lesions by rat PI-TP correlated with inability of the mammalian protein to stably associate with yeast Golgi membranes and was not a result of rat PI-TP stabilizing the endogenous sec14ts gene product. These collective data demonstrate that while the in vitro PI-TP activity of SEC14p clearly reflects some functional in vivo property of SEC14p, the PI-TP activity is not the sole essential activity of SEC14p. Those data further identify an efficient Golgi targeting capability as a likely essential feature of SEC14p function in vivo. Finally, the data suggest that stable association of SEC14p with yeast Golgi membranes is not a simple function of its lipid-binding properties, indicate that the amino-terminal 129 SEC14p residues are sufficient to direct a catalytically inactive form of rat PI-TP to the Golgi and provide the first evidence to indicate that a mammalian PI-TP can stimulate Golgi secretory function in vivo.

Specific sequences found in resident proteins of the endoplasmic reticulum (ER) and Golgi complex are believed to direct retention of these proteins in the appropriate compartment of the exocytotic pathway. Such “retention signals” have been identified at the carboxy-termini of both lumenal and membrane-bound ER resident proteins (reviewed by Pelham, 1991). We have been studying the targeting of Golgi membrane proteins using the E1 glycoprotein from the avian infectious bronchitis virus (IBV) as a model protein. This protein is targeted to cis Golgi membranes when expressed from cDNA in animal cells (Machamer et al, 1990). We previously determined that the first of the three membrane-spanning domains of the El protein contained Golgi targeting information (Machamer and Rose, 1987; Swift and Machamer, 1991). This first membrane-spanning domain (“m1”) can retain a plasma membrane protein (the vesicular stomatitis virus G protein) in the Golgi complex when inserted in place of the normal transmembrane domain. The chimeric protein (“Gm1”) is retained in the early Golgi complex suggesting that the ml domain is necessary and sufficient for the targeting of the IBV El protein to the cis Golgi complex (Swift and Machamer, 1991).

Alternative mRNA splicing of αi2, a heterotrimeric G protein α subunit, has been shown to produce an additional protein, termed sαi2. In the sαi2 splice variant, 35 novel amino acids replace the normal C-terminal 24 amino acids of αi2. Whereas αi2 is found predominantly at cellular plasma membranes, sαi2 has been localized to intracellular Golgi membranes, and the unique 35 amino acids of sαi2 have been suggested to constitute a specific targeting signal. This paper proposes and examines an alternative hypothesis: disruption of the normal C-terminus of αi2 produces an unstable protein that fails to localize to plasma membranes. sαi2 is poorly expressed upon transfection of cultured cells; however, radiolabeling indicated that αi2 and sαi2 undergo myristoylation, a co-translational modification, equally well suggesting that protein stability rather than translation is affected. Indeed, pulse-chase analysis indicates that sαi2 is more rapidly degraded compared to αi2. Co-expression of βγ rescues PM localization and increases expression of sαi2. In addition, αi2A327S, a mutant previously shown to be unstable and defective in guanine-nucleotide binding, and αi2(1–331), in which the C-terminal 24 amino acids of αi2 are deleted, show a similar pattern of subcellular localization as sαi2 (i.e., intracellular membranes rather than plasma membranes). Finally, sαi2 displays a propensity to localize to potential aggresome-like structures. Thus, instead of the novel C-terminus of sαi2 functioning as a specific Golgi targeting signal, the results presented here indicate that the disruption of the normal C-terminus of αi2 causes mislocalization and rapid degradation of sαi2.

Intracellular transport in the secretory and endocytic pathways involves the regulated assembly of cytoplasmic coat proteins on appropriate donor membranes. ARF proteins, constituting a family of small GTPases, are crucial for the membrane recruitment of coat proteins such as COP I and the adaptor complexes AP-1, AP-3 and AP-4. ARFs cycle between a GDP-bound, cytosolic form and a GTP-bound, active form that is associated with membranes. The ARF GTP–GDP cycle is regulated by GDP–GTP exchange factors (GEFs) that load ARFs with GTP and by GTPase-activating proteins (GAPs) that induce hydrolysis of GTP bound to ARFs, but the mechanistic details of this regulation remain sketchy. GGAs (Golgi-associated, γ-adaptin homologous, ARF-interacting proteins) constitute a recently discovered family of conserved proteins that associate with membranes of the trans-Golgi network (TGN) and are implicated in regulating ARF-dependent trafficking events 1xThe GGAs promote ARF-dependent recruitment of clathrin to the TGN. Puertollano, R. et al. Cell. 2001; 105: 93–102Abstract | Full Text | Full Text PDF | PubMed | Scopus (185)See all References, 2xA family of proteins with γ-adaptin and VHS domains that facilitate trafficking between the trans-Golgi network and the vacuole/lysosome. Hirst, J. et al. J. Cell Biol. 2000; 149: 67–79Crossref | PubMed | Scopus (242)See all References, 3xA selective transport route from Golgi to late endosomes that requires the yeast GGA proteins. Black, M. and Pelham, H. J. Cell Biol. 2000; 151: 587–600Crossref | PubMed | Scopus (110)See all References. In yeast, deletion of both the genes encoding GGAs results in missorting of the vacuolar enzyme carboxypeptidase Y and of the yeast syntaxin Pep12p, and a defective vacuolar morphology phenotype 2xA family of proteins with γ-adaptin and VHS domains that facilitate trafficking between the trans-Golgi network and the vacuole/lysosome. Hirst, J. et al. J. Cell Biol. 2000; 149: 67–79Crossref | PubMed | Scopus (242)See all References, 3xA selective transport route from Golgi to late endosomes that requires the yeast GGA proteins. Black, M. and Pelham, H. J. Cell Biol. 2000; 151: 587–600Crossref | PubMed | Scopus (110)See all References.Mammalian and yeast GGAs have a modular structure, comprising a VHS, GAT, hinge and γ-adaptin ear domain. The GAT domain of the GGAs binds directly to the GTP-bound form of several members of the ARF family and is sufficient to target a GFP reporter protein to the Golgi complex. The VHS domain is implicated in interactions with vesicle cargo. Puertollano et al. have now performed a structure–function analysis of the GAT and hinge domains of GGAs, focusing on human GGA3 1xThe GGAs promote ARF-dependent recruitment of clathrin to the TGN. Puertollano, R. et al. Cell. 2001; 105: 93–102Abstract | Full Text | Full Text PDF | PubMed | Scopus (185)See all References1. They identify crucial residues in a highly conserved stretch of the GAT domain required for the Golgi targeting and ARF-binding activities. Most interestingly, the GAT domain of GGA3 can inhibit the GTPase activity of ARF1 induced by recombinant GAPs in vitro (ASAP1 or ARF GAP1). In line with this observation, overexpression of the GAT domain stabilizes Golgi-bound ARF1–GTP on Golgi membranes in vivo 1xThe GGAs promote ARF-dependent recruitment of clathrin to the TGN. Puertollano, R. et al. Cell. 2001; 105: 93–102Abstract | Full Text | Full Text PDF | PubMed | Scopus (185)See all References1. Puertollano et al. show that GGAs can bind to clathrin in vitro through their hinge domain 1xThe GGAs promote ARF-dependent recruitment of clathrin to the TGN. Puertollano, R. et al. Cell. 2001; 105: 93–102Abstract | Full Text | Full Text PDF | PubMed | Scopus (185)See all References1. Binding of clathrin to liposomes is enhanced by full-length recombinant His6-GGA1 if these binding reactions additionally contain GTP-bound ARF, whereas GDP–ARF has no effect. This suggests that GGAs might promote recruitment of clathrin to membranes in cells. Indeed, expression of a Myc-tagged full-length GGA1 construct increases the staining for clathrin in the trans-Golgi complex, whereas a truncated GGA1 just containing the VHS and GAT domains results in a strong decrease in clathrin staining in the Golgi region. Thus, the GGAs could function to link clathrin to membrane-bound ARF–GTP.GGAs and GAPs might compete for interaction with ARF1–GTP, and binding of GGAs to ARF–GTP might allow for enough time for recruitment of the coat protein clathrin. Expression of a GGA1 VHS–GAT domain construct causes a striking accumulation of the cation-independent mannose 6-phosphate receptor at the TGN, but had no effect on the transferrin receptor 1xThe GGAs promote ARF-dependent recruitment of clathrin to the TGN. Puertollano, R. et al. Cell. 2001; 105: 93–102Abstract | Full Text | Full Text PDF | PubMed | Scopus (185)See all References1. This is another hint as to the transport step(s) that the GGAs mediate. It will be interesting to watch the tale of this intriguing family of proteins unfold further in the future.

Glucose transport is essential for the synthesis of lactose, the major carbohydrate and osmotic constituent of human milk, in the Golgi of mammary epithelial cells. GLUT1, the mammary gland glucose transporter, is targeted to Golgi in mouse mammary epithelial cells treated with prolactin and dexamethasone. Our purpose was to explore GLUT1 targeting in lactating mice and to determine the effect of premature weaning, at 18 d of life, on GLUT1 targeting in mammary gland. Light mitochondrial pellets from mammary glands were subjected to iodixanol density gradient centrifugation; low density fractions (1.05-1.06 g/cm3) were found to be enriched in Golgi. Western blot analysis with highly specific GLUT1 antibody, directed against the C-terminus, revealed the presence of the normally observed approximately 45 kD form of GLUT1, and also showed a signal at approximately 70 kD. Both forms were enriched in low density fractions. This was also seen after 3 h of weaning. However, after 5 h, levels of GLUT1 and the 70 kD form were selectively decreased in the low density fractions. Also, the total cellular content of the 70 kD form decreased, although GLUT1 content did not change. Returning pups to the mother for 5 h after a 5 h wean restores enrichment of the Golgi by GLUT1. The results of returning pups for 15 h after a 5 h wean, including the reappearance of the 70 kD form, were indistinguishable from the results of normally lactating mothers. We conclude that premature weaning causes a transformation of glucose transporter targeting in the lactating mouse mammary gland. During lactation, there is Golgi enrichment by GLUT1 and by a higher MW form recognized by the GLUT1 antibody. Weaning causes a reversible, transitional state that involves loss of the 70 kD form and the loss of preferential Golgi targeting of GLUT1. By diminishing the availability of free glucose within the Golgi for lactose synthesis, this provides a mechanism that reduces the rate of milk production.

International audience; The correct folding, heterodimerization and trafficking of Gn/Gc envelope glycoproteins of Rift Valley fever virus, RVFV (Bunyaviridae and Phlebovirus genus) are essential for Golgi assembly and budding of viral particles. The Gn and Gc carboxy-terminus contain a Golgi targeting and an ER-retrieval signal, respectively. We generated RVFV-like particles with mutations in the cytosolic tails of Gn or Gc and identified regions important for release of infectious particles. The role of specific amino-acids in these regions was further investigated by creating recombinant mutant viruses by reverse-genetics. Residues outside the suspected Golgi targeting motif, i.e. the di-lysine K29-K30 motif and the N43, R44 and I46 residues of the Gn cytosolic domain, appeared important for Golgi localization and RNP packaging. Concerning the Gc tail, replacement of K2 or K3 in the di-lysine motif, had a drastic impact on Gn trafficking and induced an important organelle redistribution and cell remodeling, greatly affecting particle formation and release.

Post-translational modification with myristoyl or prenyl groups is essential for membrane association of many small GTPases in the Ras superfamily. Two recent papers show that, rather than myristoylation, amino-terminal acetylation of the Arf-like protein Arl3p is required for Golgi targeting via an interaction with an integral membrane protein called Sys1.

The Rho-family small GTPase Cdc42 localizes at plasma membrane and Golgi complex and aside from protrusion and migration operates in vesicle trafficking, endo- and exocytosis as well as establishment and/or maintenance of cell polarity. The formin family members FMNL2 and -3 are actin assembly factors established to regulate cell edge protrusion during migration and invasion. Here we report these formins to additionally accumulate and function at the Golgi apparatus. As opposed to lamellipodia, Golgi targeting of these proteins required both their N-terminal myristoylation and the interaction with Cdc42. Moreover, Golgi association of FMNL2 or -3 induced a phalloidin-detectable actin meshwork around the Golgi. Importantly, functional interference with FMNL2/3 formins by RNAi or CRISPR/Cas9-mediated gene deletion invariably induced Golgi fragmentation in different cell lines. Furthermore, absence of these proteins led to enlargement of endosomes as well as defective maturation and/or sorting into late endosomes and lysosomes. In line with Cdc42 - recently established to regulate anterograde transport through the Golgi by cargo sorting and carrier formation - FMNL2/3 depletion also affected anterograde trafficking of VSV-G from the Golgi to the plasma membrane. Our data thus link FMNL2/3 formins to actin assembly-dependent functions of Cdc42 in anterograde transport through the Golgi apparatus.