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Epstein-Barr virus (EBV) replicates its genome in the nucleus and undergoes tegumentation and envelopment in the cytoplasm. We are interested in how the single-stranded DNA binding protein BALF2, which executes its function and distributes predominantly in the nucleus, is packaged into the tegument of virions. At the mid-stage of virus replication in epithelial TW01-EBV cells, a small pool of BALF2 colocalizes with tegument protein BBLF1, BGLF4 protein kinase, and the cis -Golgi marker GM130 at the perinuclear viral assembly compartment (AC). A possible nuclear localization signal (NLS) between amino acids 1100 and 1128 (C29), which contains positive charged amino acid 1113 RRKRR 1117 , is able to promote yellow fluorescent protein (YFP)-LacZ into the nucleus. In addition, BALF2 interacts with the nucleocapsid-associated protein BVRF1, suggesting that BALF2 may be transported into the cytoplasm with nucleocapsids in a nuclear egress complex (NEC)-dependent manner. A group of proteins involved in intracellular transport were identified to interact with BALF2 in a proteomic analysis. Among them, the small GTPase Rab1A functioning in bi-directional trafficking at the ER-Golgi interface is also a tegument component. In reactivated TW01-EBV cells, BALF2 colocalizes with Rab1A in the cytoplasmic AC. Expression of dominant-negative GFP-Rab1A(N124I) diminished the accumulation of BALF2 in the AC, coupling with attenuation of gp350/220 glycosylation. Virion release was significantly downregulated by expressing dominant-negative GFP-Rab1A(N124I). Overall, the subcellular distribution of BALF2 is regulated through its complex interaction with various proteins. Rab1 activity is required for proper gp350/220 glycosylation and the maturation of EBV. IMPORTANCE Upon EBV lytic reactivation, the virus-encoded DNA replication machinery functions in the nucleus, while the newly synthesized DNA is encapsidated and transported to the cytoplasm for final virus assembly. The single-stranded DNA binding protein BALF2 executing functions within the nucleus was also identified in the tegument layer of mature virions. Here, we studied the functional domain of BALF2 that contributes to the nuclear targeting and used a proteomic approach to identify novel BALF2-interacting cellular proteins that may contribute to virion morphogenesis. The GTPase Rab1, a master regulator of anterograde and retrograde endoplasmic reticulum (ER)-Golgi trafficking, colocalizes with BALF2 in the juxtanuclear concave region at the midstage of EBV reactivation. Rab1 activity is required for BALF2 targeting to the cytoplasmic assembly compartment (AC) and for gp350/220 targeting to cis -Golgi for proper glycosylation and virion release. Our study hints that EBV hijacks the bi-directional ER-Golgi trafficking machinery to complete virus assembly.

After their assembly by budding into the lumen of the intermediate compartment (IC) at the endoplasmic reticulum (ER)-Golgi interface, coronaviruses (CoVs) are released from their host cells following a pathway that remains poorly understood. The traditional view that CoV exit occurs via the constitutive secretory route has recently been questioned by studies suggesting that this process involves unconventional secretion. Here, using the avian infectious bronchitis virus (IBV) as a well-established model virus, we have applied confocal microscopy to investigate the pathway of CoV egress from epithelial Vero cells. We report a novel effect of IBV infection on cellular endomembranes, namely, the compaction of the pericentrosomal endocytic recycling compartment (ERC) defined by the GTPase Rab11, which coincides with the previously described Golgi fragmentation, as well as virus release. Despite Golgi disassembly, the IC elements containing the major IBV membrane protein (M)-which mostly associates with newly formed virus particles-maintain their close spatial connection with the Rab11-positive endocytic recycling system. Moreover, partial colocalization of the M protein with Rab11 was observed, whereas M displayed negligible overlap with LAMP-1, indicating that IBV egress does not occur via late endosomes or lysosomes. Synchronization of virus release using temperature-shift protocols was accompanied by increased colocalization of M and Rab11 in vesicular and vacuolar structures in the pericentrosomal region and at the cell periphery, most likely representing IBV-containing transport carriers. In conclusion, these results add CoVs to the growing list of viruses exploiting the endocytic recycling apparatus defined by Rab11 for their assembly and/or release., (© 2022. The Author(s).)

There has been considerable recent interest in the life cycle of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), the causative agent of the Covid-19 pandemic. Practically every step in CoV replication-from cell attachment and uptake via genome replication and expression to virion assembly has been considered as a specific event that potentially could be targeted by existing or novel drugs. Interference with cellular egress of progeny viruses could also be adopted as a possible therapeutic strategy; however, the situation is complicated by the fact that there is no broad consensus on how CoVs find their way out of their host cells. The viral nucleocapsid, consisting of the genomic RNA complexed with nucleocapsid proteins obtains a membrane envelope during virus budding into the lumen of the intermediate compartment (IC) at the endoplasmic reticulum (ER)-Golgi interface. From here, several alternative routes for CoV extracellular release have been proposed. Strikingly, recent studies have shown that CoV infection leads to the disassembly of the Golgi ribbon and the mobilization of host cell compartments and protein machineries that are known to promote Golgi-independent trafficking to the cell surface. Here, we discuss the life cycle of CoVs with a special focus on different possible pathways for virus egress., (© 2022 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Coronaviruses (CoVs) assemble by budding into the lumen of the intermediate compartment (IC) at the endoplasmic reticulum (ER)-Golgi interface. However, why CoVs have chosen the IC as their intracellular site of assembly and how progeny viruses are delivered from this compartment to the extracellular space has remained unclear. Here we address these enigmatic late events of the CoV life cycle in light of recently described properties of the IC. Of particular interest are the emerging spatial and functional connections between IC elements and recycling endosomes (REs), defined by the GTPases Rab1 and Rab11, respectively. The establishment of IC-RE links at the cell periphery, around the centrosome and evidently also at the noncompact zones of the Golgi ribbon indicates that-besides traditional ER-Golgi communication-the IC also promotes a secretory process that bypasses the Golgi stacks, but involves its direct connection with the endocytic recycling system. The initial confinement of CoVs to the lumen of IC-derived large transport carriers and their preferential absence from Golgi stacks is consistent with the idea that they exit cells following such an unconventional route. In fact, CoVs may share this pathway with other intracellularly budding viruses, lipoproteins, procollagen, and/or protein aggregates experimentally introduced into the IC lumen.

N-alpha-acetyltransferase 80 (NAA80) was recently demonstrated to acetylate the N-terminus of actin, with NAA80 knockout cells showing actin cytoskeleton-related phenotypes, such as increased formation of membrane protrusions and accelerated migration. Here we report that NAA80 knockout cells additionally display fragmentation of the Golgi apparatus. We further employed rescue assays to demonstrate that this phenotype is connected to the ability of NAA80 to modify actin. Thus, re-expression of NAA80, which leads to re-establishment of actin's N-terminal acetyl group, rescued the Golgi fragmentation, whereas a catalytic dead NAA80 mutant could neither restore actin Nt-acetylation nor Golgi structure. The Golgi phenotype of NAA80 KO cells was shared by both migrating and non-migrating cells and live-cell imaging indicated increased Golgi dynamics in migrating NAA80 KO cells. Finally, we detected a drastic increase in the amount of F-actin in cells lacking NAA80, suggesting a causal relationship between this effect and the observed re-organization of Golgi structure. The findings further underscore the importance of actin Nt-acetylation and provide novel insight into its cellular roles, suggesting a mechanistic link between actin modification state and Golgi organization., Competing Interests: Declaration of competing interests The authors declare no competing or financial interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

A characteristic feature of vertebrate cells is a Golgi ribbon consisting of multiple cisternal stacks connected into a single-copy organelle next to the centrosome. Despite numerous studies, the mechanisms that link the stacks together and the functional significance of ribbon formation remain poorly understood. Nevertheless, these questions are of considerable interest, since there is increasing evidence that Golgi fragmentation - the unlinking of the stacks in the ribbon - is intimately connected not only to normal physiological processes, such as cell division and migration, but also to pathological states, including neurodegeneration and cancer. Challenging a commonly held view that ribbon architecture involves the formation of homotypic tubular bridges between the Golgi stacks, we present an alternative model, based on direct interaction between the biosynthetic (pre-Golgi) and endocytic (post-Golgi) membrane networks and their connection with the centrosome. We propose that the central domains of these permanent pre- and post-Golgi networks function together in the biogenesis and maintenance of the more transient Golgi stacks, and thereby establish "linker compartments" that dynamically join the stacks together. This model provides insight into the reversible fragmentation of the Golgi ribbon that takes place in dividing and migrating cells and its regulation along a cell surface - Golgi - centrosome axis. Moreover, it helps to understand transport pathways that either traverse or bypass the Golgi stacks and the positioning of the Golgi apparatus in differentiated neuronal, epithelial, and muscle cells.

Despite its discovery more than three decades ago and well-established role in protein sorting and trafficking in the early secretory pathway, the intermediate compartment (IC) has remained enigmatic. The prevailing view is that the IC evolved as a specialized organelle to mediate long-distance endoplasmic reticulum (ER)-Golgi communication in metazoan cells, but is lacking in other eukaryotes, such as plants and fungi. However, this distinction is difficult to reconcile with the high conservation of the core machineries that regulate early secretory trafficking from yeast to man. Also, it has remained unclear whether the pleiomorphic IC components-vacuoles, tubules and vesicles-represent transient transport carriers or building blocks of a permanent pre-Golgi organelle. Interestingly, recent studies have revealed that the IC maintains its compositional, structural and spatial properties throughout the cell cycle, supporting a model that combines the dynamic and stable aspects of the organelle. Moreover, the IC has been assigned novel functions, such as cell signaling, Golgi-independent trafficking and autophagy. The emerging permanent nature of the IC and its connections with the centrosome and the endocytic recycling system encourage reconsideration of its relationship with the Golgi ribbon, role in Golgi biogenesis and ubiquitous presence in eukaryotic cells.

Background: Annexin A2 (AnxA2) is a multifunctional protein involved in endocytosis, exocytosis, membrane domain organisation, actin remodelling, signal transduction, protein assembly, transcription and mRNA transport, as well as DNA replication and repair., Scope of Review: The current knowledge of the role of phosphorylation in the functional regulation of AnxA2 is reviewed. To provide a more comprehensive treatment of this topic, we also address in depth the phosphorylation process in general and discuss its possible conformational effects. Furthermore, we discuss the apparent limitations of the methods used to investigate phosphoproteins, as exemplified by the study of AnxA2., Major Conclusions: AnxA2 is subjected to complex regulation by post-translational modifications affecting its cellular functions, with Ser11, Ser25 and Tyr23 representing important phosphorylation sites. Thus, Ser phosphorylation of AnxA2 is involved in the recruitment and docking of secretory granules, the regulation of its association with S100A10, and sequestration of perinuclear, translationally inactive mRNP complexes. By contrast, Tyr phosphorylation of AnxA2 regulates its role in actin dynamics and increases its association with endosomal compartments. Modification of its three main phosphorylation sites is not sufficient to discriminate between its numerous functions. Thus, fine-tuning of AnxA2 function is mediated by the joint action of several post-translational modifications., General Significance: AnxA2 participates in malignant cell transformation, and its overexpression and/or phosphorylation is associated with cancer progression and metastasis. Thus, tight regulation of AnxA2 function is an integral aspect of cellular homeostasis. The presence of AnxA2 in cancer cell-derived exosomes, as well as the potential regulation of exosomal AnxA2 by phosphorylation or other PTMs, are topics of great interest., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Tyrosine hydroxylase (TH) catalyzes the conversion of l-tyrosine into l-DOPA, which is the rate-limiting step in the synthesis of catecholamines, such as dopamine, in dopaminergergic neurons. Low dopamine levels and death of the dopaminergic neurons are hallmarks of Parkinson's disease (PD), where α-synuclein is also a key player. TH is highly regulated, notably by phosphorylation of several Ser/Thr residues in the N-terminal tail. However, the functional role of TH phosphorylation at the Ser-31 site (THSer(P)-31) remains unclear. Here, we report that THSer(P)-31 co-distributes with the Golgi complex and synaptic-like vesicles in rat and human dopaminergic cells. We also found that the TH microsomal fraction content decreases after inhibition of cyclin-dependent kinase 5 (Cdk5) and ERK1/2. The cellular distribution of an overexpressed phospho-null mutant, TH1-S31A, was restricted to the soma of neuroblastoma cells, with decreased association with the microsomal fraction, whereas a phospho-mimic mutant, TH1-S31E, was distributed throughout the soma and neurites. TH1-S31E associated with vesicular monoamine transporter 2 (VMAT2) and α-synuclein in neuroblastoma cells, and endogenous THSer(P)-31 was detected in VMAT2- and α-synuclein-immunoprecipitated mouse brain samples. Microtubule disruption or co-transfection with α-synuclein A53T, a PD-associated mutation, caused TH1-S31E accumulation in the cell soma. Our results indicate that Ser-31 phosphorylation may regulate TH subcellular localization by enabling its transport along microtubules, notably toward the projection terminals. These findings disclose a new mechanism of TH regulation by phosphorylation and reveal its interaction with key players in PD, opening up new research avenues for better understanding dopamine synthesis in physiological and pathological states., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Mammalian phenylalanine hydroxylase (PAH) has a potential allosteric regulatory binding site for l-phenylalanine (l-Phe), in addition to its catalytic site. This arrangement is supported by a crystal structure of a homodimeric truncated form of the regulatory domain of human PAH (hPAH-RD 1-118/19-118 ) [Patel D et al . (2016) Sci Rep doi: 10.1038/srep23748]. In this study, a fusion protein of the domain (MBP-(pep Xa )-hPAH-RD 1-120 ) was overexpressed and recovered in a metastable and soluble state, which allowed the isolation of a dimeric and a monomeric fusion protein. When cleaved from MBP, hPAH-RD forms aggregates which are stereospecifically inhibited by l-Phe (> 95%) at low physiological concentrations. Aggregation of the cleaved dimer of the mutant form hPAH-G46S-RD was not inhibited by l-Phe, which is compatible with structurally/conformationally changed βαββαβ ACT domain folds in the mutant.

Various post-translational modifications (PTMs) regulate the localisation and function of the multifunctional protein Annexin A2 (AnxA2). In addition to its various tasks as a cytoskeletal- and membrane-associated protein, AnxA2 can function as a trans -acting protein binding to cis -acting sequences of specific mRNAs. In the present study, we have examined the role of Ser25 phosphorylation in subcellular localisation of AnxA2 and its interaction with mRNP complexes. Subcellular fractionation and confocal microscopy of rat neuroendocrine PC12 cells showed that Ser25-phosphorylated AnxA2 (pSer25AnxA2) is absent from the nucleus and mainly localised to the perinuclear region, evidently associating with both membranes and cytoskeletal elements. Perinuclear targeting of AnxA2 was abolished by inhibition of protein kinase C activity, which resulted in cortical enrichment of the protein. Although oligo(dT)-affinity purification of mRNAs revealed that pSer25AnxA2 associates with nonpolysomal, translationally inactive mRNP complexes, it displayed only partial overlap with a marker of P-bodies. The phosphorylated protein is present as high-molecular-mass forms, indicating that it contains additional covalent PTMs, apparently triggered by its Ser25 phosphorylation. The subcellular distributions of these forms clearly differ from the main form of AnxA2 and are also distinct from that of Tyr23-phosphorylated AnxA2. Immunoprecipitation verified that these high-molecular-mass forms are due to ubiquitination and/or sumoylation. Moreover, these results indicate that Ser25 phosphorylation and ubiquitin/SUMO1 conjugation of AnxA2 promote its association with nonpolysomal mRNAs, providing evidence of a possible mechanism to sequester a subpopulation of mRNAs in a translationally inactive and transport competent form at a distinct subcellular localisation.

Two conserved Rab GTPases, Rab1 and Rab2, play important roles in biosynthetic-secretory trafficking between the endoplasmic reticulum (ER) and the Golgi apparatus in mammalian cells. Both are expressed as two isoforms that regulate anterograde transport via the intermediate compartment (IC) to the Golgi, but are also required for transport in the retrograde direction. Moreover, Rab1 has been implicated in the formation of autophagosomes. Rab1 and Rab2 have numerous effectors or partners that function in membrane tethering, but also have other roles. These include the coiled-coil proteins p115, GM130, giantin, golgin-84, and GMAP-210, as well as the multisubunit COG (conserved oligomeric Golgi) and TRAPP (transport protein particle) tethering complexes. TRAPP also acts as the GTP exchange factor (GEF) in the activation of Rab1. According to the traditional view of the IC elements as motile, transient structures, the functions of the Rabs could take place at the two ends of the ER-Golgi itinerary, i.e., at ER exit sites (ERES) and/or cis-Golgi. However, there is considerable evidence for their specific association with the IC, including its recently identified pericentrosomal domain (pcIC), where many of the effectors turn out to be present, thus being able to exert their functions at the pre-Golgi level. The IC localization of these proteins is of particular interest based on the imaging of Rab1 dynamics, indicating that the IC is a stable organelle that bidirectionally communicates with the ER and Golgi, and is functionally linked to the endosomal system via the pcIC.

Annexin A2 (AnxA2) is a multi-functional and -compartmental protein whose subcellular localisation and functions are tightly regulated by its post-translational modifications. AnxA2 and its Tyr23-phosphorylated form (pTyr23AnxA2) are involved in malignant cell transformation, metastasis and angiogenesis. Here, we show that H2O2 exerts rapid, simultaneous and opposite effects on the Tyr23 phosphorylation status of AnxA2 in two distinct compartments of rat pheochromocytoma (PC12) cells. Reactive oxygen species induce dephosphorylation of pTyr23AnxA2 located in the PML bodies of the nucleus, whereas AnxA2 associated with F-actin at the cell cortex is Tyr23 phosphorylated. The H2O2-induced responses in both compartments are transient and the pTyr23AnxA2 accumulating at the cell cortex is subsequently incorporated into vesicles and then released to the extracellular space. Blocking nuclear export by leptomycin B does not affect the nuclear pool of pTyr23AnxA2, but increases the amount of total AnxA2 in this compartment, indicating that the protein might have several functions in the nucleus. These results suggest that Tyr23 phosphorylation can regulate the function of AnxA2 at distinct subcellular sites., (© 2016. Published by The Company of Biologists Ltd.)

Purpose: Congenital stromal corneal dystrophy (CSCD) is an autosomal dominant condition with clouding of the cornea due to stromal opacities. It is caused by mutations in the decorin gene (DCN) leading to the expression of a truncated form of decorin. In an attempt to replicate this condition in mice, a knock-in mouse strain, 952delT Dcn, was created., Methods: Mice were constructed by targeted mutation. Sequencing of genomic DNA confirmed correct genotype. Mouse and human corneas, including corneas from patients with CSCD, and primary keratocyte cultures were subjected to Western blot analysis, transmission electron microscopy, and immunofluorescence microscopy., Results: Histologically, the mice did not show any organ pathology. Corneas were clear, and the electron-lucent deposits observed in CSCD were not present. Furthermore, while nearly equivalent amounts of normal and truncated decorin are present in CSCD corneas, truncated decorin was hardly detectable in the mouse corneas. By immunofluorescence analysis of corneas from 952delT Dcn homozygous mice, decorin was found only in keratocytes. In primary cultures of mouse corneal explants, truncated decorin was retained intracellularly in contrast with human corneal explants where truncated decorin was exported into the culture medium. Immunofluorescence analysis revealed that native mouse decorin localized to the Golgi complex, whereas the truncated decorin accumulated in the endoplasmic reticulum (ER)., Conclusions: The ER retention of truncated decorin may explain why the mouse corneas remained clear. The consequences of the decorin mutation are different in mice and humans, and 952delT Dcn knock-in mice are therefore not a suitable model for CSCD.

Maturity-onset diabetes of the young, type 8 (MODY8) is characterized by a syndrome of autosomal dominantly inherited diabetes and exocrine pancreatic dysfunction. It is caused by deletion mutations in the last exon of the carboxyl ester lipase (CEL) gene, resulting in a CEL protein with increased tendency to aggregate. In this study we investigated the intracellular distribution of the wild type (WT) and mutant (MUT) CEL proteins in cellular models. We found that both CEL-WT and CEL-MUT were secreted via the endoplasmic reticulum and Golgi compartments. However, their subcellular distributions differed, as only CEL-MUT was observed as an aggregate at the cell surface and inside large cytoplasmic vacuoles. Many of the vacuoles were identified as components of the endosomal system, and after its secretion, the mutant CEL protein was re-internalized, transported to the lysosomes, and degraded. Internalization of CEL-MUT also led to reduced viability of pancreatic acinar and beta cells. These findings may have implications for the understanding of how the acinar-specific CEL-MUT protein causes both exocrine and endocrine pancreatic disease., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Annexin A2 (AnxA2) interacts with numerous ligands, including calcium, lipids, mRNAs and intracellular and extracellular proteins. Different post-translational modifications participate in the discrimination of the functions of AnxA2 by modulating its ligand interactions. Here, phospho-mimicking mutants (AnxA2-S25E and AnxA2-S25D) were employed to investigate the effects of Ser25 phosphorylation on the structure and function of AnxA2 by using AnxA2-S25A as a control. The overall α-helical structure of AnxA2 is not affected by the mutations, since the thermal stabilities and aggregation tendencies of the mutants differ only slightly from the wild-type (wt) protein. Unlike wt AnxA2, all mutants bind the anxA2 3' untranslated region and β-γ-G-actin with high affinity in a Ca(2+)-independent manner. AnxA2-S25E is not targeted to the nucleus in transfected PC12 cells. In vitro phosphorylation of AnxA2 by protein kinase C increases its affinity to mRNA and inhibits its nuclear localisation, in accordance with the data obtained with the phospho-mimicking mutants. Ca(2+)-dependent binding of wt AnxA2 to phosphatidylinositol, phosphatidylinositol-3-phosphate, phosphatidylinositol-4-phosphate and phosphatidylinositol-5-phosphate, as well as weaker but still Ca(2+)-dependent binding to phosphatidylserine and phosphatidylinositol-3,5-bisphosphate, was demonstrated by a protein-lipid overlay assay, whereas binding of AnxA2 to these lipids, as well as its binding to liposomes, is inhibited by the Ser25 mutations. Thus, introduction of a modification (mutation or phosphorylation) at Ser25 appears to induce a conformational change leading to increased accessibility of the mRNA- and G-actin-binding sites in domain IV independent of Ca(2+) levels, while the Ca(2+)-dependent binding of AnxA2 to phospholipids is attenuated., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Studies carried out during the last 2 decades have dramatically increased our knowledge of the pathways and mechanisms of intracellular membrane traffic, most recently due to the developments in light microscopy and in vivo imaging of fluorescent fusion proteins. These studies have also revealed that certain molecules do not behave according to the classical transportation rules first documented in cell biology textbooks in the 1980s and 1990s. Initially, unconventional mechanisms of secretion that do not involve passage of cargo through the stacked Golgi cisternae were thought to confer on cells the ability to discard excess amounts of protein products. With time, however, more physiological mechanisms and roles have been proposed for an increasing number of secretory processes that bypass the Golgi apparatus.

As mammalian cells prepare for mitosis, the Golgi ribbon is first unlinked into its constituent stacks and then transformed into spindle-associated, pleiomorphic membrane clusters in a process that remains enigmatic. Also, it remains unclear whether Golgi inheritance involves the incorporation of Golgi enzymes into a pool of coat protein I (COPI) vesicles, or their COPI-independent transfer to the endoplasmic reticulum (ER). Based on the observation that the intermediate compartment (IC) at the ER-Golgi boundary is connected to the centrosome, we examined its mitotic fate and possible role in Golgi breakdown. The use of multiple imaging techniques and markers revealed that the IC elements persist during the M phase, maintain their compositional and structural properties and remain associated with the mitotic spindle, forming circular arrays at the spindle poles. At G2/M transition, the movement of the pericentrosomal domain of the IC (pcIC) to the cell centre and its expansion coincide with the unlinking of the Golgi ribbon. At prophase, coupled to centrosome separation, the pcIC divides together with recycling endosomes, providing novel landmarks for mitotic entry. We provide evidence that the permanent IC elements function as way stations during the COPI-dependent dispersal of Golgi components at prometa- and metaphase, indicating that they correspond to the previously described Golgi clusters. In addition, they continue to communicate with the vesicular 'Golgi haze' and thus are likely to provide templates for Golgi reassembly. These results implicate the IC in mitotic Golgi inheritance, resulting in a model that integrates key features of the two previously proposed pathways.

CEL-maturity onset diabetes of the young (MODY), diabetes with pancreatic lipomatosis and exocrine dysfunction, is due to dominant frameshift mutations in the acinar cell carboxyl ester lipase gene (CEL). As Cel knock-out mice do not express the phenotype and the mutant protein has an altered and intrinsically disordered tandem repeat domain, we hypothesized that the disease mechanism might involve a negative effect of the mutant protein. In silico analysis showed that the pI of the tandem repeat was markedly increased from pH 3.3 in wild-type (WT) to 11.8 in mutant (MUT) human CEL. By stably overexpressing CEL-WT and CEL-MUT in HEK293 cells, we found similar glycosylation, ubiquitination, constitutive secretion, and quality control of the two proteins. The CEL-MUT protein demonstrated, however, a high propensity to form aggregates found intracellularly and extracellularly. Different physicochemical properties of the intrinsically disordered tandem repeat domains of WT and MUT proteins may contribute to different short and long range interactions with the globular core domain and other macromolecules, including cell membranes. Thus, we propose that CEL-MODY is a protein misfolding disease caused by a negative gain-of-function effect of the mutant proteins in pancreatic tissues.

The missense mutation pG46S in the regulatory (R) domain of human phenylalanine hydroxylase (hPAH), associated with a severe form of phenylketonuria, generates a misfolded protein which is rapidly degraded on expression in HEK293 cells. When overexpressed as a MBP-G46S fusion protein, soluble and fully active tetrameric/dimeric forms are assembled and recovered in a metastable conformational state. When MBP is cleaved off, G46S undergoes a conformational change and self-associates with a lag phase and an autocatalytic growth phase (tetramers≫dimers), as determined by light scattering. The self-association is controlled by pH, ionic strength, temperature, protein concentration and the phosphorylation state of Ser16; the net charge of the protein being a main modulator of the process. A superstoichiometric amount of WT dimers revealed a 2-fold enhancement of the rate of G46S dimer self-association. Electron microscopy demonstrates the formation of higher-order oligomers and linear polymers of variable length, partly as a branching network, and partly as individual long and twisted fibrils (diameter ~145-300Å). The heat-shock proteins Hsp70/Hsp40, Hsp90 and a proposed pharmacological PAH chaperone (3-amino-2-benzyl-7-nitro-4-(2-quinolyl)-1,2-dihydroisoquinolin-1-one) partly inhibit the self-association process. Our data indicate that the G46S mutation results in a N-terminal extension of α-helix 1 which perturbs the wild-type α-β sandwich motif in the R-domain and promotes new intermolecular contacts, self-association and non-amyloid fibril formation. The metastable conformational state of G46S as a MBP fusion protein, and its self-association propensity when released from MBP, may represent a model system for the study of other hPAH missense mutations characterized by misfolded proteins., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Phenylketonuria (PKU), the most common inborn error of metabolism, is caused by dysfunction of the liver enzyme phenylalanine hydroxylase (PAH), with more than 550 PAH gene mutations identified to date. A large number of these mutations result in mutant forms of the enzyme displaying reduced stability, increased propensity to aggregate, and accelerated in cellulo degradation. Loss or reduction of human PAH activity results in hyperphenylalaninemia (HPA) which, if untreated, results in severe mental retardation and impaired cognitive development. Until now, strict low phenylalanine diet has been the most effective therapy, but as a protein misfolding disease PKU is a good candidate for treatment by natural/chemical/pharmacological chaperones. The natural cofactor of human PAH, (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH(4)), has already been approved for oral treatment of HPA, giving a positive response in mild forms of the disease showing considerable residual enzymatic activity. In the case of the most severe forms of PKU, ongoing studies with chemical and pharmacological chaperones to rescue misfolded mutant proteins from aggregation and degradation are providing promising results. The PKU mutation G46S is associated with a severe form of the disease, resulting in an aggregation-prone protein. The human PAH mutant G46S is rapidly degraded in the cellular environment and, in vitro (upon removal of its stabilizing fusion partner maltose binding protein (MBP)) self-associates to form higher-order oligomers/fibrils. Here, we present an in vitro experimental model system to study the modulation of G46S aggregation by chemical/pharmacological chaperones, which may represent a useful approach to study the rescue of other severe PKU mutations by chemical/pharmacological chaperones., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The intermediate compartment (IC) between the endoplasmic reticulum (ER) and the Golgi apparatus appears to constitute an autonomous organelle composed of spatially and functionally distinct, but interconnected, vacuolar and tubular subdomains. In mammalian cells the IC network is stably anchored at the cell center, communicating directly with the endocytic pathway via a pericentrosomal membrane system (PCMS). This finding suggests that the secretory pathway divides at the level of the IC, which functions as a sorting station both in Golgi-dependent and -independent trafficking. The tubular subdomain of the IC is capable of expansion in accordance with its proposed biosynthetic functions such as cholesterol synthesis.

Because the functional borders of the intermediate compartment (IC) are not well defined, the spatial map of the transport machineries operating between the endoplasmic reticulum (ER) and the Golgi apparatus remains incomplete. Our previous studies showed that the IC consists of interconnected vacuolar and tubular parts with specific roles in pre-Golgi trafficking. Here, using live cell imaging, we demonstrate that the tubules containing the GTPase Rab1A create a long-lived membrane compartment around the centrosome. Separation of this pericentrosomal domain of the IC from the Golgi ribbon, due to centrosome motility, revealed that it contains a distinct pool of COPI coats and acts as a temperature-sensitive way station in post-ER trafficking. However, unlike the Golgi, the pericentrosomal IC resists the disassembly of COPI coats by brefeldin A, maintaining its juxtaposition with the endocytic recycling compartment, and operation as the focal point of a dynamic tubular network that extends to the cell periphery. These results provide novel insight into the compartmental organization of the secretory pathway and Golgi biogenesis. Moreover, they reveal a direct functional connection between the IC and the endosomal system, which evidently contributes to unconventional transport of the cystic fibrosis transmembrane conductance regulator to the cell surface.

Cholesterol, certain lipids, membrane-bound and soluble proteins, as well as viruses that are synthesized in the endoplasmic reticulum (ER), reach the plasma membrane (PM) via non-classical pathway(s) that remain poorly understood. Typical for this transport is (i) its insensitivity to brefeldin A (BFA), which dissociates selected coat complexes from membranes, resulting in the disassembly of the Golgi apparatus; (ii) its rapid kinetics as compared to the classical secretory pathway; and (iii) its role in the trafficking of lipid raft components. Based on results showing that the intermediate compartment (IC) at the ER-Golgi boundary constitutes a stable tubular network that maintains its dynamics in the presence of BFA, we propose that two bidirectional Golgi-bypass pathways to the PM exist, a direct route from early IC elements, and another, reminiscent of the yeast secretory pathway, from late IC elements via the endosomal system. These pathways have implications for the organization of the secretory processes in different cell types.

A prerequisite for understanding the cellular functions of an unknown protein is the establishment of its subcellular localization. As increasing numbers of novel proteins of the biosynthetic pathway are currently being identified, accessible new methods are required to facilitate their localization. Differentiating rat pheochromocytoma (PC12) cells reorganize their biosynthetic membrane compartments as they develop neurite-like processes. The authors recently showed that polarization of these cells involves the expansion of the intermediate compartment (IC) between the rough endoplasmic reticulum (RER) and the Golgi apparatus. Tubules emerging from the vacuolar parts of the IC move to the developing neurites accumulating in their growth cones, whereas the vacuoles, like RER and Golgi, remain in the cell body. Thus, polarized PC12 cells enhance the resolution for immunofluorescence microscopic mapping of protein localization in the early biosynthetic pathway. The authors also describe here a rapid cell fractionation protocol employing velocity sedimentation in iodixanol gradients that allows one-step separation of the pre-Golgi vacuoles, tubules, and RER.

In higher eukaryotic cells pleiomorphic compartments composed of vacuoles, tubules and vesicles move from the endoplasmic reticulum (ER) and the plasma membrane to the cell center, operating in early biosynthetic trafficking and endocytosis, respectively. Besides transporting cargo to the Golgi apparatus and lysosomes, a major task of these compartments is to promote extensive membrane recycling. The endocytic membrane system is traditionally divided into early (sorting) endosomes, late endosomes and the endocytic recycling compartment (ERC). Recent studies on the intermediate compartment (IC) between the ER and the Golgi apparatus suggest that it also consists of peripheral ("early") and centralized ("late") structures, as well as a third component, designated here as the biosynthetic recycling compartment (BRC). We propose that the ERC and the BRC exist as long-lived "mirror compartments" at the cell center that also share the ability to expand and become mobilized during cell activation. These considerations emphasize the functional symmetry of endomembrane compartments, which provides a basis for the membrane rearrangements taking place during cell division, polarization, and differentiation.

The function of the pre-Golgi intermediate compartment (IC) and its relationship with the endoplasmic reticulum (ER) and Golgi remain only partially understood. Here, we report striking segregation of IC domains in polarized PC12 cells that develop neurite-like processes. Differentiation involves expansion of the IC and movement of Rab1-containing tubules to the growth cones of the neurites, whereas p58- and COPI-positive IC elements, like rough ER and Golgi, remain in the cell body. Exclusion of Rab1 effectors p115 and GM130 from the neurites further indicated that the centrifugal, Rab1-mediated pathway has functions that are not directly related to ER-to-Golgi trafficking. Disassembly of COPI coats did not affect this pathway but resulted in missorting of p58 to the neurites. Live cell imaging showed that green fluorescent protein (GFP)-Rab1A-containing IC elements move bidirectionally both within the neurites and cell bodies, interconnecting different ER exit sites and the cis-Golgi region. Moreover, in nonpolarized cells GFP-Rab1A-positive tubules moved centrifugally towards the cell cortex. Hydroxymethylglutaryl-CoA reductase, the key enzyme of cholesterol biosynthesis, colocalized with slowly sedimenting, Rab1-enriched membranes when the IC subdomains were separated by velocity sedimentation. These results reveal a novel pathway directly connecting the IC with the cell periphery and suggest that this Rab1-mediated pathway is linked to the dynamics of smooth ER.

In the secretory pathway, the forward (anterograde) membrane flow is compensated by retrograde transport of proteins and lipids. Membrane recycling is required for the maintenance of organelle homeostasis and the re-use of components of the transport machineries for the generation of new transport intermediates. However, the molecular mechanisms and other cellular functions of retrograde traffic are still poorly understood. In recent years, a multitude of protein factors that function in the secretory pathway have been discovered, most of them originally suggested to play a role in forward trafficking. However, in many cases subsequent studies have revealed that these proteins participate (also) in retrograde traffic. It is likely that this shift will continue, reflecting the fact that the two pathways are intimately connected.

We have studied the localization of functional components of cellular Ca2+ transport and storage and the effects of thapsigargin (TG), a specific inhibitor of the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), with respect to the p58-containing pre-Golgi intermediate compartment (IC). The depletion of Ca2+ stores in normal rat kidney (NRK) cells by TG abolished the retention of the KDEL-containing, Ca2+-binding, luminal ER chaperones GRP94/endoplasmin and GRP78/BiP, and resulted in the appearance of the proteins in the culture medium before inducing their synthesis. Immunolocalization of GRP94 in TG-treated cells showed that the protein was transported to the Golgi complex and, in parallel, the KDEL receptor was redistributed from the Golgi to p58-positive IC structures, but was not transported further to the ER. Similarly, p58 that normally cycles between the ER, IC, and cis-Golgi, was largely depleted from the cell periphery and arrested in large-sized IC elements and numerous vesicles or buds in the Golgi region, showing that TG selectively blocks its recycling from the IC back to the ER. Importantly, cell fractionation analyses and confocal fluorescence microscopy provided evidence that the IC elements in unperturbed cells contain SERCA and a considerable pool of GRP94. Thus, the observed effects of TG on protein retention and recycling can be explained by a change in the luminal Ca2+ concentration of the IC. Moreover, the compositional properties of the IC elements suggest that they participate in intracellular Ca2+ storage.

We have studied the structural and functional properties of the pre-Golgi intermediate compartment (IC) in normal rat kidney cells using analytical cell fractionation with p58 as the principal marker. The sedimentation profile (sediterm) of p58, obtained by analytical differential centrifugation, revealed in steady-state cells the presence of two main populations of IC elements whose average sedimentation coefficients, s(H)=1150+/-58S ('heavy') and s(L)=158+/-8S ('light'), differed from the s-values obtained for elements of the rough and smooth endoplasmic reticulum. High resolution analysis of these subpopulations in equilibrium density gradients further revealed that the large difference in their s-values was mainly due to particle size. The 'light' particle population contained the bulk of COPI and COPII coats, and redistribution of p58 to these particles was observed in transport-arrested cells, showing that the two types of elements are also compositionally distinct and have functional counterparts in intact cells. Using a specific antibody against the 16 kDa proteolipid subunit of the vacuolar H(+)-ATPase, an enrichment of the V(o )domain of the ATPase was observed in the p58-positive IC elements. Interestingly, these elements could contain both COPI and COPII coats and their density distribution was markedly affected by GTP(&ggr;)S. Together with morphological observations, these results demonstrate that, in addition to clusters of small tubules and vesicles, the IC also consists of large-sized structures and corroborate the proposal that the IC elements contain an active vacuolar H(+)-ATPase.

The effect of the vacuolar H+-ATPase inhibitor bafilomycin A1 (Baf A1) on the localization of pre-Golgi intermediate compartment (IC) and Golgi marker proteins was used to study the role of acidification in the function of early secretory compartments. Baf A1 inhibited both brefeldin A- and nocodazole-induced retrograde transport of Golgi proteins to the endoplasmic reticulum (ER), whereas anterograde ER-to-Golgi transport remained largely unaffected. Furthermore, p58/ERGIC-53, which normally cycles between the ER, IC, and cis-Golgi, was arrested in pre-Golgi tubules and vacuoles, and the number of p58-positive approximately 80-nm Golgi (coatomer protein I) vesicles was reduced, suggesting that the drug inhibits the retrieval of the protein from post-ER compartments. In parallel, redistribution of beta-coatomer protein from the Golgi to peripheral pre-Golgi structures took place. The small GTPase rab1p was detected in short pre-Golgi tubules in control cells and was efficiently recruited to the tubules accumulating in the presence of Baf A1. In contrast, these tubules showed no enrichment of newly synthesized, anterogradely transported proteins, indicating that they participate in retrograde transport. These results suggest that the pre-Golgi structures contain an active H+-ATPase that regulates retrograde transport at the ER-Golgi boundary. Interestingly, although Baf A1 had distinct effects on peripheral pre-Golgi structures, only more central, p58-containing elements accumulated detectable amounts of 3-(2, 4-dinitroanilino)-3'-amino-N-methyldipropylamine (DAMP), a marker for acidic compartments, raising the possibility that the lumenal pH of the pre-Golgi structures gradually changes in parallel with their translocation to the Golgi region.

Previous studies have shown that caffeine treatment at 20 degrees C causes the intermediate compartment protein p58 to redistribute from the Golgi region without affecting the localization of the Golgi stack protein mannosidase II (J. Jäntti, E. Kuismanen, J. Cell Biol. 120, 1321-1335 (1993). Here we have dissected further the effect of caffeine on transport of Golgi and intermediate compartment proteins from the cell periphery to the perinuclear Golgi region. To accumulate proteins in the peripheral membranes, BHK-21 cells were treated with brefeldin A to redistribute marker proteins towards the ER. Following BFA wash-out and subsequent incubation at 15 degrees C, p58, the coat protein beta-COP, and Man II were all localized in the peripheral 15 degrees C-intermediates. When the cells were shifted from 15 degrees C to 20 degrees C all the proteins were recentralized to the Golgi region. However, if the temperature shift was carried out in the presence of 10 mM caffeine, p58 and beta-COP maintained their peripheral localization, whereas Man II was transported to the Golgi region. The results indicate that caffeine at 20 degrees C does not block the centralization of Man II from peripheral sites to the central Golgi region. Therefore, its effect on ER to Golgi transport appears to be manifested specifically at ER exit. Furthermore, our results indicate that segregation of intermediate compartment and Golgi stack proteins can occur at the level of the peripheral 15 degrees C-intermediates. Immunoelectron microscopic localization of p58 and Man II showed that these peripheral intermediates consisted of tubules and small stacks of cisternae. Within the tubular intermediates both p58 and Man II appeared to segregate to membrane subdomains. Finally, examination of serial and thick sections support the idea that the stacked structures can be generated from tubular intermediates.

The organization of membrane trafficking between endoplasmic reticulum and Golgi within multinucleated muscle fibers was analyzed. We found that markers for the compartment involved in endoplasmic reticulum to Golgi trafficking exhibited perinuclear as well as interfibrillar localization. Furthermore, these markers showed prominent colocalization with microtubules. To analyze membrane trafficking, we followed the temperature-controlled transport of the G protein of the mutant vesicular stomatitis virus, tsO45, in isolated myofibers. Perinuclear and cross-striated staining were seen at 39 degrees C, while at 15 degrees C a diffuse staining component appeared along a subset of interfibrillar microtubules. At 20 degrees C, bright Golgi spots were seen to be associated with microtubules that appeared as circumnuclear rings and longitudinal bundles. Beneath the motor end plate, however, the organization of the Golgi elements and microtubules was found to be distinctive. Retrograde trafficking induced by brefeldin A resulted in the disappearance of the Golgi spots throughout the myofibers and the appearance of staining along microtubules. Thus, interfibrillar membranes seem to be active in protein export, and trafficking between endoplasmic reticulum and Golgi elements occurred throughout the myofibers. The results suggest that microtubules served as tracks for the two-way trafficking between the endoplasmic reticulum and the Golgi compartment.

An abundant 58-kDa (p58) homodimeric and hexameric microsomal membrane protein has been biochemically characterized and localized to tubulo-vesicular elements at the endoplasmic reticulum-Golgi interface and the cis-Golgi cisternae in pancreatic acinar cells (Lahtinen, U., Dahllöf, B., and Saraste, J. (1992) J. Cell Sci. 103, 321-333). Here we report the purification of p58 by two-dimensional gel electrophoresis, and the cloning and sequencing of the rat and part of the Xenopus laevis cDNAs. The rat cDNA encodes a 517-amino acid protein having a putative signal sequence, a transmembrane domain close to the C terminus and a short cytoplasmic tail. The C-terminal tail contains a double-lysine motif (KKFF), known to mediate retrieval of proteins from the Golgi back to the endoplasmic reticulum. The rat p58 sequence was found to be 89% identical with those of ERGIC-53 and MR60, two previously identified human membrane proteins. Strong homology with the frog sequence was also observed indicating high evolutionary conservation. Overexpression of c-Myc-tagged p58 resulted in accumulation of the protein both in the endoplasmic reticulum and in an apparently enlarged Golgi complex, as well as its leakage to the plasma membrane. Immunolocalization using antibodies raised against a lumenal peptide stained the total cellular pool of p58, while anti-tail peptide antibodies detected p58 only in a restricted Golgi region. This suggests that the C-terminal tail of p58 located in the endoplasmic reticulum and transport intermediates is hidden, but becomes exposed when the protein reaches the Golgi complex.

A number of cellular proteins and viral spike proteins are cleaved at a basic recognition sequence. To characterize the membrane traffic step at which this proteolysis occurs we have studied the intracellular processing site of Semliki Forest virus (SFV) spike precursor p62 in BHK21 cells. The p62 is endoproteolytically cleaved at a tetrabasic Arg-His-Arg-Arg recognition sequence. Previously, it has been shown that the SFV p62 remains uncleaved when accumulated to the trans-Golgi network (TGN/20 degrees C block site). We show here that exit from the trans-Golgi is required for the cleavage of p62. Proteolytic processing was inhibited in synchronized assays when the 20 degrees C transport block was released in the presence of brefeldin A, energy inhibitors (azide and deoxyglucose; carbonyl cyanide m-chlorophenylhydrazone, CCCP) or an effector of trimeric G proteins, AlFn. Endocytosed antibodies against the SFV spike glycoproteins or antibodies against a peptide corresponding to the enzymatically active motif of furin inhibited cleavage of p62 at a post-TGN location. The results indicate a post-TGN communication step between exocytic and endocytic elements. Kinetic experiments suggested that this communication may involve an early compartment of the endocytic pathway.

The aim of this study was to investigate the antimigratory and antiinvasive potential of vincristine sulfate (VCR) on human glioma cells and to analyze whether phenytoin (5,5-diphenylhydantoin; DPH) might act synergistically with VCR. Vincristine affects the cytoplasmic microtubules; DPH has been reported to enhance VCR cytotoxicity in murine cells. In two human glioma cell lines, GaMG and D-37MG, we found VCR to reduce monolayer growth and colony formation in a dose-dependent fashion at concentrations of 10 ng/ml and above. Phenytoin increased the cytotoxic and cytostatic effects of VCR in monolayer cells but not in spheroids. Multicellular spheroids were used to investigate directional migration. A coculture system of GaMG and D-37MG spheroids with fetal rat brain aggregates was used to analyze and quantify tumor cell invasion. A dose-dependent inhibition of migration and invasion by VCR was observed in both cell lines without further enhancement by DPH. Immunofluorescence microscopy with antibodies against alpha-tubulin revealed dose-dependent morphological alterations in the microtubules when the cells were exposed to VCR but not after incubation with DPH. Based on the combination of standardized in vitro model systems currently in use and the present data, the authors strongly suggest that VCR inhibits migration and invasion of human glioma cells. This is not altered by DPH, which inhibits cell proliferation in combination with VCR.