The life cycle and enigmatic egress of coronaviruses.

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.

Evidence for the role of Rab11-positive recycling endosomes as intermediates in coronavirus egress from epithelial cells.

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.

N-terminal acetylation of actin by NAA80 is essential for structural integrity of the Golgi apparatus.

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.

Phosphorylation at serine 31 targets tyrosine hydroxylase to vesicles for transport along microtubules.

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.

Reactive oxygen species exert opposite effects on Tyr23 phosphorylation of the nuclear and cortical pools of annexin A2.

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.

Intermediate compartment (IC): from pre-Golgi vacuoles to a semi-autonomous membrane system.

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.

Protein phosphorylation and its role in the regulation of Annexin A2 function.

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.

Endocytosis of secreted carboxyl ester lipase in a syndrome of diabetes and pancreatic exocrine dysfunction.

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.

Division of the intermediate compartment at the onset of mitosis provides a mechanism for Golgi inheritance.

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.

Arrivals and departures at the plasma membrane: direct and indirect transport routes.

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.

Subcellular Distribution of BALF2 and the Role of Rab1 in the Formation of Epstein-Barr Virus Cytoplasmic Assembly Compartment and Virion Release.

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 1113RRKRR1117, 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.

Diabetes and pancreatic exocrine dysfunction due to mutations in the carboxyl ester lipase gene-maturity onset diabetes of the young (CEL-MODY): a protein misfolding disease.

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.

Phenylketonuria as a protein misfolding disease: The mutation pG46S in phenylalanine hydroxylase promotes self-association and fibril formation.

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-ß 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.

The G46S-hPAH mutant protein: a model to study the rescue of aggregation-prone PKU mutations by chaperones.

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.

Retrograde traffic in the biosynthetic-secretory route: pathways and machinery.

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.

Assembly and Cellular Exit of Coronaviruses: Hijacking an Unconventional Secretory Pathway from the Pre-Golgi Intermediate Compartment via the Golgi Ribbon to the Extracellular Space.

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.

Functional symmetry of endomembranes.

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.

Rab1 defines a novel pathway connecting the pre-Golgi intermediate compartment with the cell periphery.

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.

A New Look at the Functional Organization of the Golgi Ribbon.

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.

Use of polarized PC12 cells to monitor protein localization in the early biosynthetic pathway.

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.