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The GalNAc-type O-glycoproteome is orchestrated by a large family of polypeptide GalNAc-transferase isoenzymes (GalNAc-Ts) with partially overlapping contributions to the O-glycoproteome besides distinct nonredundant functions. Increasing evidence indicates that individual GalNAc-Ts co-regulate and fine-tune specific protein functions in health and disease, and deficiencies in individual GALNT genes underlie congenital diseases with distinct phenotypes. Studies of GalNAc-T specificities have mainly been performed with in vitro enzyme assays using short peptide substrates, but recently quantitative differential O-glycoproteomics of isogenic cells with and without GALNT genes has enabled a more unbiased exploration of the nonredundant contributions of individual GalNAc-Ts. Both approaches suggest that fairly small subsets of O-glycosites are nonredundantly regulated by specific GalNAc-Ts, but how these isoenzymes orchestrate regulation among competing redundant substrates is unclear. To explore this, here we developed isogenic cell model systems with Tet-On inducible expression of two GalNAc-T genes, GALNT2 and GALNT11, in a knockout background in HEK293 cells. Using quantitative O-glycoproteomics with tandem-mass-tag (TMT) labeling, we found that isoform-specific glycosites are glycosylated in a dose-dependent manner and that induction of GalNAc-T2 or -T11 produces discrete glycosylation effects without affecting the major part of the O-glycoproteome. These results support previous findings indicating that individual GalNAc-T isoenzymes can serve in fine-tuned regulation of distinct protein functions.

The surface glycoprotein (GP) of Ebola virus causes many of the virus's pathogenic effects, including a dramatic loss of endothelial cell adhesion associated with widespread hemorrhaging during infection. Although the GP-mediated deadhesion depends on its extracellular mucin-like domain, it is unknown whether any, or all, of this domain's densely clustered O-glycosylation sites are required. It is also unknown whether any of the 20 distinct polypeptide GalNAc-transferases (ppGalNAc-Ts) that initiate mucin-type O-glycosylation in human cells are functionally required. Here, using HEK293 cell lines lacking specific glycosylation enzymes, we demonstrate that GP requires extended O-glycans to exert its deadhesion effect. We also identified ppGalNAc-T1 as largely required for the GP-mediated adhesion defects. Despite its profound effect on GP function, the absence of ppGalNAc-T1 only modestly reduced the O-glycan mass of GP, indicating that even small changes in the bulky glycodomain can cause loss of GP function. Indeed, protein-mapping studies identified a small segment of the mucin-like domain critical for function and revealed that mutation of five glycan acceptor sites within this segment are sufficient to abrogate GP function. Together, these results argue against a mechanism of Ebola GP–induced cell detachment that depends solely on ectodomain bulkiness and identify a single host-derived glycosylation enzyme, ppGalNAc-T1, as a potential target for therapeutic intervention against Ebola virus disease.

International audience; The retrograde transport inhibitor Retro-2 has a protective effect on cells and in mice against Shiga-like toxins and ricin. Retro-2 causes toxin accumulation in early endosomes and relocalization of the Golgi SNARE protein syntaxin-5 to the endoplasmic reticulum. The molecular mechanisms by which this is achieved remain unknown. Here, we show that Retro-2 targets the endoplasmic reticulum exit site component Sec16A, affecting anterograde transport of syntaxin-5 from the endoplasmic reticulum to the Golgi. The formation of canonical SNARE complexes involving syntaxin-5 is not affected in Retro-2-treated cells. By contrast, the interaction of syntaxin-5 with a newly discovered binding partner, the retrograde trafficking chaperone GPP130, is abolished, and we show that GPP130 must indeed bind to syntaxin-5 to drive Shiga toxin transport from the endosomes to the Golgi. We therefore identify Sec16A as a druggable target and provide evidence for a non-SNARE function for syntaxin-5 in interaction with GPP130.

By binding and directing the cycling Golgi protein GPP130 to lysosomes, the sorting receptor sortilin mediates the manganese-induced GPP130 down-regulation that protects against Shiga toxicosis., Elevated, nontoxic doses of manganese (Mn) protect against Shiga toxin-1–induced cell death via down-regulation of GPP130, a cycling Golgi membrane protein that serves as an endosome-to-Golgi trafficking receptor for the toxin. Mn binds to GPP130 in the Golgi and causes GPP130 to oligomerize/aggregate, and the complexes are diverted to lysosomes. In fact, based on experiments using the self-interacting FM domain, it appears generally true that aggregation of a Golgi protein leads to its lysosomal degradation. How such oligomers are selectively sorted out of the Golgi is unknown. Here we provide evidence that Mn-induced exit of GPP130 from the trans-Golgi network (TGN) toward lysosomes is mediated by the sorting receptor sortilin interacting with the lumenal stem domain of GPP130. In contrast, FM-induced lysosomal trafficking of the Golgi protein galactosyltransferase was sortilin independent and occurred even in the absence of its native lumenal domain. Thus sortilin-dependent as well as sortilin-independent sorting mechanisms target aggregated Golgi membrane proteins for lysosomal degradation.

Oligomerization or homotypic clustering diverts Golgi membrane proteins into the canonical GGA1/clathrin-dependent Golgi-to-lysosome pathway revealing the presence of cellular quality control that could be useful for therapies designed to down-regulate specific proteins in the secretory pathway., Manganese protects cells against forms of Shiga toxin by down-regulating the cycling Golgi protein GPP130. Down-regulation occurs when Mn binding causes GPP130 to oligomerize and traffic to lysosomes. To determine how GPP130 is redirected to lysosomes, we tested the role of GGA1 and clathrin, which mediate sorting in the canonical Golgi-to-lysosome pathway. GPP130 oligomerization was induced using either Mn or a self-interacting version of the FKBP domain. Inhibition of GGA1 or clathrin specifically blocked GPP130 redistribution, suggesting recognition of the aggregated GPP130 by the GGA1/clathrin-sorting complex. Unexpectedly, however, GPP130’s cytoplasmic domain was not required, and redistribution also occurred after removal of GPP130 sequences needed for its normal cycling. Therefore, to test whether aggregate recognition might be a general phenomenon rather than one involving a specific GPP130 determinant, we induced homo-oligomerization of two unrelated Golgi-targeted constructs using the FKBP strategy. These were targeted to the cis- and trans-Golgi, respectively, using domains from mannosidase-1 and galactosyltransferase. Significantly, upon oligomerization, each redistributed to peripheral punctae and was degraded. This occurred in the absence of detectable UPR activation. These findings suggest the unexpected presence of quality control in the Golgi that recognizes aggregated Golgi proteins and targets them for degradation in lysosomes.

The mammalian Golgi reassembly stacking protein (GRASP) proteins are Golgi-localized homotypic membrane tethers that organize Golgi stacks into a long, contiguous ribbon-like structure. It is unknown how GRASPs undergo trans pairing given that cis interactions between the proteins in the plane of the membrane are intrinsically favored. To test the hypothesis that myristoylation of the self-interacting GRASP domain restricts its orientation on the membrane to favor trans pairing, we established an in vitro assay that recapitulates GRASP-dependent membrane tethering and used neutron reflection under similar conditions to determine the orientation of the GRASP domain. In vivo, the membrane association of GRASP proteins is conferred by the simultaneous insertion of an N-terminal myristic acid and binding to a Golgi-associated binding partner. In our assay, the latter contact was replaced using a C-terminal hexa-His moiety, which bound to Ni2+-conjugated lipids incorporated into a substrate-supported bilayer lipid membrane. Nonmyristoylated protein lacked a fixed orientation on the membrane and inefficiently tethered liposomes. In contrast, myristoylated GRASP promoted tethering and exhibited a unique membrane complex. Thus, myristoylation restricts the membrane orientation of the GRASP domain favoring interactions in trans for membrane tethering. Published version

Use of photoinactivation, cisternae-specific fluorescence recovery, and high-resolution microscopy shows that the membrane tethers GRASP65 and GRASP55 on early and late Golgi membranes, respectively, are critical to the specific, homotypic fusion of the membranes on which they reside., Homotypic membrane tethering by the Golgi reassembly and stacking proteins (GRASPs) is required for the lateral linkage of mammalian Golgi ministacks into a ribbon-like membrane network. Although GRASP65 and GRASP55 are specifically localized to cis and medial/trans cisternae, respectively, it is unknown whether each GRASP mediates cisternae-specific tethering and whether such specificity is necessary for Golgi compartmentalization. Here each GRASP was tagged with KillerRed (KR), expressed in HeLa cells, and inhibited by 1-min exposure to light. Significantly, inactivation of either GRASP unlinked the Golgi ribbon, and the immediate effect of GRASP65-KR inactivation was a loss of cis- rather than trans-Golgi integrity, whereas inactivation of GRASP55-KR first affected the trans- and not the cis-Golgi. Thus each GRASP appears to play a direct and cisternae-specific role in linking ministacks into a continuous membrane network. To test the consequence of loss of cisternae-specific tethering, we generated Golgi membranes with a single GRASP on all cisternae. Remarkably, the membranes exhibited the full connectivity of wild-type Golgi ribbons but were decompartmentalized and defective in glycan processing. Thus the GRASP isoforms specifically link analogous cisternae to ensure Golgi compartmentalization and proper processing.

Mucin-type O-glycosylation occurring in the Golgi apparatus is an important protein posttranslational modification initiated by up to 20 GalNAc-transferase isozymes with largely distinct substrate specificities. Regulation of this enzyme family affects a vast array of proteins transiting the secretory pathway and misregulation causes human diseases. Here we describe the use of protein-based fluorescence sensors that traffic in the secretory pathway to monitor GalNAc-transferase activity in living cells. The sensors can either be “pan” or isozyme specific.

Originally identified as Golgi stacking factors in vitro, the Golgi reassembly stacking protein (GRASP) family has been shown to act as membrane tethers with multiple cellular roles. As an update to previous comprehensive reviews of the GRASP family (Giuliani et al., 2011; Vinke et al., 2011; Jarvela and Linstedt, 2012), we outline here the latest findings concerning their diverse roles. New insights into the mechanics of GRASP-mediated tethering come from recent crystal structures. The models of how GRASP65 and GRASP55 tether membranes relate directly to their role in Golgi ribbon formation in mammalian cells and the unlinking of the ribbon at the onset of mitosis. However, it is also clear that GRASPs act outside the Golgi with roles at the ER and ER exit sites (ERES). Furthermore, the proteins of this family display other roles upon cellular stress, especially in mediating unconventional secretion of both transmembrane proteins (Golgi bypass) and cytoplasmic proteins (through secretory autophagosomes).

Protein O-glycosylation is important in numerous processes including the regulation of proteolytic processing sites by O-glycan masking in select newly synthesized proteins. To investigate O-glycan-mediated masking using an assay amenable to large-scale screens, we generated a fluorescent biosensor with an O-glycosylation site situated to mask a furin cleavage site. The sensor is activated when O-glycosylation fails to occur because furin cleavage releases a blocking domain allowing dye binding to a fluorogen activating protein. Thus, by design, glycosylation should block furin from activating the sensor only if it occurs first, which is predicted by the conventional view of Golgi organization. Indeed, and in contrast to the recently proposed rapid partitioning model, the sensor was non-fluorescent under normal conditions but became fluorescent when the Golgi complex was decompartmentalized. To test the utility of the sensor as a screening tool, cells expressing the sensor were exposed to a known inhibitor of O-glycosylation extension or siRNAs targeting factors known to alter glycosylation efficiency. These conditions activated the sensor substantiating its potential in identifying new inhibitors and cellular factors related to protein O-glycosylation. In summary, these findings confirm sequential processing in the Golgi, establish a new tool for studying the regulation of proteolytic processing by O-glycosylation, and demonstrate the sensor's potential usefulness for future screening projects.

Shunning Shiga Infection with bacteria harboring Shiga toxin is responsible for more than 1 million deaths annually worldwide. Antidotes for the toxin are not available, and treatment with antibiotics is contraindicated because it increases the risk of toxin release (from dead bacteria) and leads to severe forms of the disease. Manganese is an essential nutrient and its toxicology in humans is well studied; it inhibits the normal trafficking of Shiga toxin in tissue culture cells. Mukhopadhyay and Linstedt (p. 332 ) found that levels of Mn 2+ that caused no deleterious effects to normal cellular processes nevertheless altered intracellular trafficking of Shiga toxin so that it was degraded in lysosomes. This conferred very high levels of protection of cultured cells against toxin-induced death and made mice completely resistant to Shiga toxin–induced paralysis and death.

P-type ATPases transport a wide array of ions, regulate diverse cellular processes, and are implicated in a number of human diseases. However, mechanisms that increase ion transport by these ubiquitous proteins are not known. SPCA1 is a P-type pump that transports Mn 2+ from the cytosol into the Golgi. We developed an intra-Golgi Mn 2+ sensor and used it to screen for mutations introduced in SPCA1, on the basis of its predicted structure, which could increase its Mn 2+ pumping activity. Remarkably, a point mutation (Q747A) predicted to increase the size of its ion permeation cavity enhanced the sensor response and a compensatory mutation restoring the cavity to its original size abolished this effect. In vivo and in vitro Mn 2+ transport assays confirmed the hyperactivity of SPCA1-Q747A. Furthermore, increasing Golgi Mn 2+ transport by expression of SPCA1-Q747A increased cell viability upon Mn 2+ exposure, supporting the therapeutic potential of increased Mn 2+ uptake by the Golgi in the management of Mn 2+ -induced neurotoxicity.

GRASP65 links cis-Golgi cisternae via a homotypic, N-terminal PDZ interaction, and its mitotic phosphorylation disrupts this activity. Neither the identity of the PDZ ligand involved in the GRASP65 self-interaction nor the mechanism by which phosphorylation inhibits its interaction is known. Phospho-mimetic mutation of known cyclin-dependent kinase 1/cyclin B sites, all of which are in the C-terminal “regulatory domain” of the molecule, failed to block organelle tethering. However, we identified a site phosphorylated by Polo-like kinase 1 (PLK1) in the GRASP65 N-terminal domain for which mutation to aspartic acid blocked tethering and alanine substitution prevented mitotic Golgi unlinking. Further, using interaction assays, we discovered an internal PDZ ligand adjacent to the PLK phosphorylation site that was required for tethering. These results reveal the mechanism of phosphoinhibition as direct inhibition by PLK1 of the PDZ ligand underlying the GRASP65 self-interaction.

GRASP proteins share an N-terminal GRASP domain and mediate homotypic tethering of Golgi cisternae to form extended Golgi ribbons. The golgin GM130 is thought to bind the C-terminal side of the GRASP domain to recruit GRASP65 onto the Golgi whereas stable membrane association appears to also depend on anchoring of the N terminus by myristoylation. Here, we examine the nature of the GM130/GRASP65 interaction and test whether the dual membrane contacts of the GRASP domain have a role in tethering beyond membrane recruitment. GM130 was found to contain a C-terminal PDZ ligand that binds the putative groove of the second PDZ-like domain in GRASP65. To test tethering activity independent of targeting, we took advantage of a tethering assay carried out on the mitochondrial membrane in which the GRASP membrane attachment points were individually or simultaneously substituted with mitochondrially targeted transmembrane sequences. N-terminally anchored constructs tethered only if the C terminus was also anchored; and likewise, C-terminally anchored constructs tethered only if the N terminus was anchored. One explanation for the role of this dual anchoring is that it orients the GRASP domain to prevent cis interactions within the same membrane thereby favoring trans interactions between adjacent membranes. Indeed, singly anchored GRASP constructs, although nonfunctional in tethering, interacted with one another and also bound and inhibited dually anchored constructs. This work thus elucidates the GM130/GRASP65 interaction and supports a novel orientation-based model of membrane tether regulation in which dual membrane contact orients the tethering interaction interface to favor trans over cis interactions.

We propose a new active mask algorithm for the segmentation of fluorescence microscope images of punctate patterns. It combines the (a) flexibility offered by active-contour methods, (b) speed offered by multiresolution methods, (c) smoothing offered by multiscale methods, and (d) statistical modeling offered by region-growing methods into a fast and accurate segmentation tool. The framework moves from the idea of the ldquocontourrdquo to that of ldquoinside and outside,rdquo or masks, allowing for easy multidimensional segmentation. It adapts to the topology of the image through the use of multiple masks. The algorithm is almost invariant under initialization, allowing for random initialization, and uses a few easily tunable parameters. Experiments show that the active mask algorithm matches the ground truth well and outperforms the algorithm widely used in fluorescence microscopy, seeded watershed, both qualitatively, as well as quantitatively.

The Golgi apparatus is comprised of stacked cisternal membranes forming subcompartments specialized for posttranslational processing of newly synthesized secretory cargo. Recent experimental evidence indicates that the Golgi apparatus can undergo de novo biogenesis from the endoplasmic reticulum, but the mechanism by which the membranes self assemble into compartmentalized structures remains unknown. We developed a discrete-event computer simulation model to test whether two fundamental mechanisms—vesicle-coat-mediated selective concentration of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins during vesicle formation, and SNARE-mediated selective fusion of vesicles—suffice to generate and maintain compartments. Simulations verified that this minimal model is adequate for homeostasis of preestablished compartments, even in response to small perturbations, and for de novo formation of stable compartments. The model led to a novel prediction that Golgi size is in part dependent on target SNARE expression level. This prediction was supported by a demonstration that exogenous expression of the Golgi target SNARE syntaxin-5 alters Golgi size in living cells.

Under experimental conditions, the Golgi apparatus can undergo de novo biogenesis from the endoplasmic reticulum (ER), involving a rapid phase of growth followed by a return to steady state, but the mechanisms that control growth are unknown. Quantification of coat protein complex (COP) II assembly revealed a dramatic up-regulation at exit sites driven by increased levels of Golgi proteins in the ER. Analysis in a permeabilized cell assay indicated that up-regulation of COPII assembly occurred in the absence GTP hydrolysis and any cytosolic factors other than the COPII prebudding complex Sar1p–Sec23p–Sec24p. Remarkably, acting via a direct interaction with Sar1p, increased expression of the Golgi enzyme N-acetylgalactosaminyl transferase-2 induced increased COPII assembly on the ER and an overall increase in the size of the Golgi apparatus. These results suggest that direct interactions between Golgi proteins exiting the ER and COPII components regulate ER exit, providing a variable exit rate mechanism that ensures homeostasis of the Golgi apparatus.

The mammalian Golgi apparatus exists as stacks of cisternae that are laterally linked to form a continuous membrane ribbon, but neither the molecular requirements for, nor the purpose of, Golgi ribbon formation are known. Here, we demonstrate that ribbon formation is mediated by specific membrane-fusion events that occur during Golgi assembly, and require the Golgi proteins GM130 and GRASP65. Furthermore, these GM130 and GRASP65-dependent lateral cisternal-fusion reactions are necessary to achieve uniform distribution of enzymes in the Golgi ribbon. The membrane continuity created by ribbon formation facilitates optimal processing conditions in the biosynthetic pathway.

The subcompartmentalized structure of the Golgi apparatus contributes to efficient glycosylation in the secretory pathway. Subcompartmentalization driven by maturation relies primarily on constant and accurate vesicle-mediated local recycling of Golgi residents. The precision of this vesicle transport is dependent on the interplay between the key factors that mediate vesicle budding and fusion--the coat proteins and the SNARE fusion machinery. These alone, however, may not be sufficient to ensure establishment of compartments de novo, and additional regulatory mechanisms operate to modify their activity.

Functional characterization of protein interactions in mammalian systems has been hindered by the inability to perform complementation analyses in vivo . Here, we use functional replacement of the vesicle docking protein p115 to separate its essential from its nonessential interactions. p115 is required for biogenesis of the Golgi apparatus, but it is unclear whether its mechanism of action requires its golgin and/or SNARE interactions. Short interfering RNA-mediated knockdown of p115 induced extensive Golgi fragmentation and impaired secretory traffic. Reassembly of a structurally and functionally normal Golgi occurred on expression of a p115 homologue not recognized by the short interfering RNA. Strikingly, versions of p115 lacking its phosphorylation site and the golgin-binding domains also restored the Golgi apparatus in cells lacking endogenous p115. In contrast, the p115 SNARE-interacting domain was required for Golgi biogenesis. This suggests that p115 acts directly, rather than via a tether, to catalyze trans-SNARE complex formation preceding membrane fusion.

All identified basolateral sorting signals of integral membrane proteins are cytoplasmically disposed, suggesting that basolateral targeting is mediated exclusively by direct interaction with vesicle coat components. Here, we report that GPP130, a cis-Golgi protein that undergoes endosome-to-Golgi retrieval using the late endosome-bypass pathway in nonpolarized cells, cycles via the basolateral membrane in polarized MDCK cells. Significantly, the membrane-proximal lumenal domain of GPP130, which mediates GPP130 localization and trafficking in nonpolarized cells, was both necessary and sufficient for basolateral cycling in MDCK cells. The use of lumenal determinants for both basolateral cycling and endosome-to-Golgi retrieval suggests that a novel receptor-mediated mechanism operates at both the trans-Golgi network and distal sites to sort GPP130 along the late-endosome-bypass retrieval pathway in polarized cells.

The cis-Golgi protein GPP130 reversibly redistributes to endosomes upon pH disruption, but the identity of the endosomes and the involved cycling route are unknown. It is also unknown whether any other early Golgi proteins participate in this pathway. Here, we analyze GPP130 and the structurally related Golgi protein GP73. Unlike the TGN marker TGN38/46, GPP130 and GP73 colocalized in the early Golgi and redistributed to the ER after brefeldin A treatment. Nevertheless, after pH disruption by monensin, GPP130 and GP73 redistributed to endosomes containing redistributed TGN38/46, but not other endosomal markers. In common with TGN38/46, the redistribution involved transient appearance on the plasma membrane, and upon monensin washout, the proteins moved back to the Golgi along a microtubule- and PI3 kinase-independent route. Although GP73 did not associate with GPP130, its steady-state Golgi targeting was also mediated by a lumenal predicted coiled-coil stem domain. These findings indicate that at least two early Golgi proteins, each containing stem domain Golgi targeting determinants, cycle to the cell surface and back along the late endosome independent TGN38/46 pathway.

Generating loss of protein function is a powerful investigatory tool particularly if carried out on a physiologically relevant timescale in a live-cell fluorescent imaging experiment. KillerRed mediated chromophore assisted light inactivation (CALI) uses genetic encoding for specificity and light for acute inactivation that can also be spatially restricted. This unit provides protocols for setting up and carrying out properly controlled KillerRed experiments during live-cell imaging of cultured cells.

Humans express up to twenty isoforms of GalNAc-transferase (herein T1-T20) that localize to the Golgi apparatus and initiate O-glycosylation. Regulation of this enzyme family affects a vast array of proteins transiting the secretory pathway and diseases arise upon misregulation of specific isoforms. Surprisingly, molecular probes to monitor GalNAc-transferase activity are lacking and there exist no effective global or isoform-specific inhibitors. Here we describe the development of T2- and T3-isoform specific fluorescence sensors that traffic in the secretory pathway. Each sensor yielded little signal when glycosylated but was strongly activated in the absence of its glycosylation. Specificity of each sensor was assessed in HEK cells with either the T2 or T3 enzyme deleted. Although the sensors are based on specific substrates of the T2 and T3 enzymes, elements in or near the enzyme recognition sequence influenced their activity and required modification, which we carried out based on previous in vitro work. Significantly, the modified T2 and T3 sensors were activated only in cells lacking their corresponding isozymes. Thus, we have developed T2 and T3 specific sensors that will be valuable in both the study of GalNAc-transferase regulation and in high-throughput screening for potential therapeutic regulators of specific GalNAc-transferases.

Golgi inheritance during cell division involves Golgi disassembly but it remains unclear whether the breakdown product is dispersed vesicles, clusters of vesicles or a fused ER/Golgi network. Evidence against the fused ER/Golgi hypothesis was previously obtained from subcellular fractionation studies, but left concerns about the means used to obtain and disrupt mitotic cells. Here, we performed velocity gradient analysis on otherwise untreated cells shaken from plates 9 h after release from an S-phase block. In addition, we used digitonin and freeze/thaw permeabilization as alternatives to mechanical homogenization. Under each of these conditions, approximately 75% of the Golgi was recovered in a population of small vesicles that lacked detectable ER. We also used multilabel fluorescent microscopy with optical sectioning by deconvolution to compare the 3D metaphase staining pattern of endogenous Golgi and ER markers. Although both ER and Golgi staining were primarily diffuse, only the ER was excluded from the mitotic spindle region. Surprisingly, only 2% of the Golgi fluorescence was present as resolvable structures previously characterized as vesicle clusters. These were not present in the ER pattern. Significantly, a portion of the diffuse Golgi fluorescence, presumably representing dispersed 60-nm vesicles, underwent an apparent rapid aggregation with the larger Golgi structures upon treatments that impaired microtubule integrity. Therefore, mitotic Golgi appears to be in a dynamic equilibrium between clustered and free vesicles, and accurate partitioning may be facilitated by microtubule-based motors acting on the clusters to insure random and uniform distribution of the vesicles.

Inhibition of the putative coatomer protein I (COPI) vesicle tethering complex, giantin–p115–GM130, may contribute to mitotic Golgi breakdown. However, neither this, nor the role of the giantin–p115–GM130 complex in the maintenance of Golgi structure has been demonstrated in vivo. Therefore, we generated antibodies directed against the mapped binding sites in each protein of the complex and injected these into mammalian tissue culture cells. Surprisingly, the injected anti-p115 and antigiantin antibodies caused proteasome-mediated degradation of the corresponding antigens. Reduction of p115 levels below detection led to COPI-dependent Golgi fragmentation and apparent accumulation of Golgi-derived vesicles. In contrast, neither reduction of giantin below detectable levels, nor inhibition of p115 binding to GM130, had any detectable effect on Golgi structure or Golgi reassembly after cell division or brefeldin A washout. These observations indicate that inhibition of p115 can induce a mitotic-like Golgi disassembly, but its essential role in Golgi structure is independent of its Golgi-localized binding partners giantin and GM130.

Despite the potential importance of retrieval-based targeting, few Golgi cisternae-localized proteins have been demonstrated to be targeted by retrieval, and the putative retrieval signals remain unknown. Golgi phosphoprotein of 130 kDa (GPP130) is acis-Golgi protein that allows assay of retrieval-based targeting because it redistributes to endosomes upon treatment with agents that disrupt lumenal pH, and it undergoes endosome-to-Golgi retrieval upon drug removal. Analysis of chimeric molecules containing domains from GPP130 and the plasma membrane protein dipeptidylpeptidase IV indicated that GPP130 targeting information is contained entirely within its lumenal domain. Dissection of the lumenal domain indicated that a predicted coiled-coil stem domain adjacent to the transmembrane domain was both required and sufficient for pH-sensitive Golgi localization and endosome-to-Golgi retrieval. Further dissection of this stem domain revealed two noncontiguous stretches that each conferred Golgi localization separated by a stretch that conferred endosomal targeting. Importantly, in the absence of the endosomal determinant the Golgi targeting of constructs containing either or both of the Golgi determinants became insensitive to pH disruption by monensin. Because monensin blocks endosome-to-Golgi transport, the finding that the endosomal determinant confers monensin sensitivity suggests that the endosomal determinant causes GPP130 to traffic to endosomes from which it is normally retrieved. Thus, our observations identify Golgi and endosomal targeting determinants within a lumenal predicted coiled-coil domain that appear to act coordinately to mediate retrieval-based targeting of GPP130.

The role of the mitogen-activated protein kinase kinase (MKK)/extracellular-activated protein kinase (ERK) pathway in mitotic Golgi disassembly is controversial, in part because Golgi-localized targets have not been identified. We observed that Golgi reassembly stacking protein 55 (GRASP55) was phosphorylated in mitotic cells and extracts, generating a mitosis-specific phospho-epitope recognized by the MPM2 mAb. This phosphorylation was prevented by mutation of ERK consensus sites in GRASP55. GRASP55 mitotic phosphorylation was significantly reduced, both in vitro and in vivo, by treatment with U0126, a potent and specific inhibitor of MKK and thus ERK activation. Furthermore, ERK2 directly phosphorylated GRASP55 on the same residues that generated the MPM2 phospho-epitope. These results are the first demonstration of GRASP55 mitotic phosphorylation and indicate that the MKK/ERK pathway directly phosphorylates the Golgi during mitosis.

Recent evidence suggests a regulatory connection between cell volume, endoplasmic reticulum (ER) export, and stimulated Golgi-to-ER transport. To investigate the potential role of protein kinases we tested a panel of protein kinase inhibitors for their effect on these steps. One inhibitor, H89, an isoquinolinesulfonamide that is commonly used as a selective protein kinase A inhibitor, blocked both ER export and hypo-osmotic-, brefeldin A-, or nocodazole-induced Golgi-to-ER transport. In contrast, H89 did not block the constitutive ER Golgi-intermediate compartment (ERGIC)-to-ER and Golgi-to-ER traffic that underlies redistribution of ERGIC and Golgi proteins into the ER after ER export arrest. Surprisingly, other protein kinase A inhibitors, KT5720 and H8, as well as a set of protein kinase C inhibitors, had no effect on these transport processes. To test whether H89 might act at the level of either the coatomer protein (COP)I or the COPII coat protein complex we examined the localization of βCOP and Sec13 in H89-treated cells. H89 treatment led to a rapid loss of Sec13-labeled ER export sites but βCOP localization to the Golgi was unaffected. To further investigate the effect of H89 on COPII we developed a COPII recruitment assay with permeabilized cells and found that H89 potently inhibited binding of exogenous Sec13 to ER export sites. This block occurred in the presence of guanosine-5′-O-(3-thio)triphosphate, suggesting that Sec13 recruitment is inhibited at a step independent of the activation of the GTPase Sar1. These results identify a requirement for an H89-sensitive factor(s), potentially a novel protein kinase, in recruitment of COPII to ER export sites, as well as in stimulated but not constitutive Golgi-to-ER transport.

By forming a molecular tether between two membranes, p115, giantin, and GM130 may mediate multiple Golgi-related processes including vesicle transport, cisternae formation, and cisternal stacking. The tether is proposed to involve the simultaneous binding of p115 to giantin on one membrane and to GM130 on another membrane. To explore this model, we tested for the presence of the putative giantin-p115-GM130 ternary complex. We first mapped p115-binding site in giantin to a 70-amino acid coiled-coil domain at the extreme N terminus, a position that may exist up to 400 nm away from the Golgi membrane. We then generated glutathioneS-transferase (GST) fusion proteins containing either giantin's or GM130's p115 binding site and tested whether such proteins could bind p115 and GM130 or bind p115 and giantin, respectively. Unexpectedly, GST fusions containing either the giantin or the GM130 p115 binding site efficiently bound p115, but the p115 bound to GST-giantin did not bind GM130, and the p115 bound to GST-GM130 did not bind giantin. To explain this result, we mapped the giantin binding site in p115 and found that it is located at the C-terminal acidic domain, the same domain involved in binding GM130. The presence of a single binding site in p115 for giantin and GM130 was confirmed by demonstration that giantin and GM130 compete for binding to p115. These results question a simple tethering model involving a ternary giantin-p115-GM130 complex and suggest that p115-giantin and p115-GM130 interactions might mediate independent membrane tethering events.

The Golgi complex breaks down at mitosis and is subsequently reassembled in a process that involves cisternae formation and stacking. Recent studies have provided new insights into the molecular events that mediate both formation and stacking of the Golgi cisternae during post-mitotic reassembly.

Partitioning of the mammalian Golgi apparatus during cell division involves disassembly at M-phase. Despite the importance of the disassembly/reassembly pathway in Golgi biogenesis, it remains unclear whether mitotic Golgi breakdown in vivo proceeds by direct vesiculation or involves fusion with the endoplasmic reticulum (ER). To test whether mitotic Golgi is fused with the ER, we compared the distribution of ER and Golgi proteins in interphase and mitotic HeLa cells by immunofluorescence microscopy, velocity gradient fractionation, and density gradient fractionation. While mitotic ER appeared to be a fine reticulum excluded from the region containing the spindle-pole body, mitotic Golgi appeared to be dispersed small vesicles that penetrated the area containing spindle microtubules. After cell disruption, M-phase Golgi was recovered in two size classes. The major breakdown product, accounting for at least 75% of the Golgi, was a population of 60-nm vesicles that were completely separated from the ER using velocity gradient separation. The minor breakdown product was a larger, more heterogenously sized, membrane population. Double-label fluorescence analysis of these membranes indicated that this portion of mitotic Golgi also lacked detectable ER marker proteins. Therefore we conclude that the ER and Golgi remain distinct at M-phase in HeLa cells. To test whether the 60-nm vesicles might form from the ER at M-phase as the result of a two-step vesiculation pathway involving ER–Golgi fusion followed by Golgi vesicle budding, mitotic cells were generated with fused ER and Golgi by brefeldin A treatment. Upon brefeldin A removal, Golgi vesicles did not emerge from the ER. In contrast, the Golgi readily reformed from similarly treated interphase cells. We conclude that Golgi-derived vesicles remain distinct from the ER in mitotic HeLa cells, and that mitotic cells lack the capacity of interphase cells for Golgi reemergence from the ER. These experiments suggest that mitotic Golgi breakdown proceeds by direct vesiculation independent of the ER.

Manganese is specifically effective against Shiga toxin (STx) and STx1. STx2 does not bind the manganese-sensitive host cell receptor GPP130, because a histidine/asparagine pair that constitutes the binding site for GPP130 is not conserved. This reveals an unexpected and significant functional divergence in Shiga toxin evolution., Shiga toxicosis is caused by retrograde trafficking of one of three types of Shiga toxin (STx), STx, STx1, or STx2. Trafficking depends on the toxin B subunits, which for STx and STx1 are identical and bind GPP130, a manganese (Mn)-sensitive intracellular trafficking receptor. Elevated Mn down-regulates GPP130, rendering STx/STx1 harmless. Its effectiveness against STx2, however, which is a serious concern in the developed world, is not known. Here we show that Mn-induced GPP130 down-regulation fails to block STx2 trafficking. To shed light on this result, we tested the purified B subunit of STx2 for binding to GPP130 and found that it failed to interact. We then mapped residues at the interface of the GPP130-STx/STx1 complex. In GPP130, binding mapped to a seven-residue stretch in its lumenal stem domain next to the transmembrane domain. This stretch was required for STx/STx1 transport. In STx/STx1, binding mapped to a histidine–asparagine pair on a surface-exposed loop of the toxin B subunit. Significantly, these residues are not conserved in STx2, explaining the lack of effectiveness of Mn against STx2. Together our results imply that STx2 uses an evolutionarily distinct trafficking mechanism and that Mn as a potential therapy should be focused on STx/STx1 outbreaks, which account for the vast majority of cases worldwide.

Bacterial AB5 toxins are a clinically relevant class of exotoxins that include several well-known members such as Shiga, cholera, and pertussis toxins. Infections with toxin-producing bacteria cause devastating human diseases that affect millions of individuals each year and have no definitive medical treatment. The molecular targets of AB5 toxins reside in the cytosol of infected cells, and the toxins reach the cytosol by trafficking through the retrograde membrane transport pathway that avoids degradative late endosomes and lysosomes. Focusing on Shiga toxin as the archetype member, we review recent advances in understanding the molecular mechanisms involved in the retrograde trafficking of AB5 toxins and highlight how these basic science advances are leading to the development of a promising new therapeutic approach based on inhibiting toxin transport.

Acute inhibition is a powerful technique to test proteins for direct roles and order their activities in a pathway, but as a general gene-based strategy, it is mostly unavailable in mammalian systems. As a consequence, the precise roles of proteins in membrane trafficking have been difficult to assess in vivo. Here we used a strategy based on a genetically encoded fluorescent protein that generates highly localized and damaging reactive oxygen species to rapidly inactivate exit from the endoplasmic reticulum (ER) during live-cell imaging and address the long-standing question of whether the integrity of the Golgi complex depends on constant input from the ER. Light-induced blockade of ER exit immediately perturbed Golgi membranes, and surprisingly, revealed that cis-Golgi-resident proteins continuously cycle to peripheral ER-Golgi intermediate compartment (ERGIC) membranes and depend on ER exit for their return to the Golgi. These experiments demonstrate that ER exit and extensive cycling of cis-Golgi components to the cell periphery sustain the mammalian Golgi complex.

Mitotic phosphorylation of the conserved GRASP domain of GRASP65 disrupts its self-association, leading to a loss of Golgi membrane tethering, cisternal unlinking, and Golgi breakdown. Recently, the structural basis of the GRASP self-interaction was determined, yet the mechanism by which phosphorylation disrupts this activity is unknown. Here, we present the crystal structure of a GRASP phosphomimic containing an aspartic acid substitution for a serine residue (Ser-189) that in GRASP65 is phosphorylated by PLK1, causing a block in membrane tethering and Golgi ribbon formation. The structure revealed a conformational change in the GRASP internal ligand that prevented its insertion into the PDZ binding pocket, and gel filtration assays showed that this phosphomimic mutant exhibited a significant reduction in dimer formation. Interestingly, the structure also revealed an apparent propagation of conformational change from the site of phosphorylation to the shifted ligand, and alanine substitution of two residues (Glu-145 and Ser-146) at penultimate positions in this chain rescued dimer formation by the phosphomimic. These data reveal the structural basis of the phosphoinhibition of GRASP-mediated membrane tethering and provide a mechanism for its allosteric regulation.

The Golgi complex processes secretory proteins and lipids, carries out protein sorting and signaling, and supports growth and composition of the plasma membrane. Golgi complex size likely is regulated to meet the demands of each function, and this may involve differential changes of its distinct subdomains. Nevertheless, the primary size change is elongation of the Golgi ribbon-like network as occurs during Golgi complex doubling for mitosis and during differentiation involving upregulated secretion. One hypothesis states that Golgi complex size is set by the abundance of secretory cargo and Golgi complex components that, through binding vesicle coat complexes, drive vesicle coat formation to alter Golgi complex influx and efflux. Regulation of transport factors controlling Golgi membrane traffic is also observed and may control Golgi complex size, but more work is needed to directly link these events to Golgi complex size regulation, especially during differentiation of specialized cell types.

Biogenesis of the ribbon-like membrane network of the mammalian Golgi requires membrane tethering by the conserved GRASP domain in GRASP65 and GRASP55, yet the tethering mechanism is not fully understood. Here, we report the crystal structure of the GRASP55 GRASP domain, which revealed an unusual arrangement of two tandem PDZ folds that more closely resemble prokaryotic PDZ domains. Biochemical and functional data indicated that the interaction between the ligand-binding pocket of PDZ1 and an internal ligand on PDZ2 mediates the GRASP self-interaction, and structural analyses suggest that this occurs via a unique mode of internal PDZ ligand recognition. Our data uncover the structural basis for ligand specificity and provide insight into the mechanism of GRASP-dependent membrane tethering of analogous Golgi cisternae.

Manganism is a disease with no cure. This study identifies a mammalian protein with manganese-sensitive trafficking. The findings provide an important, novel example of regulated sorting under physiological conditions particularly in that a lumenal, rather than cytoplasmic, sequence confers the regulation., Manganese is an essential element that is also neurotoxic at elevated exposure. However, mechanisms regulating Mn homeostasis in mammalian cells are largely unknown. Because increases in cytosolic Mn induce rapid changes in the localization of proteins involved in regulating intracellular Mn concentrations in yeast, we were intrigued to discover that low concentrations of extracellular Mn induced rapid redistribution of the mammalian cis-Golgi glycoprotein Golgi phosphoprotein of 130 kDa (GPP130) to multivesicular bodies. GPP130 was subsequently degraded in lysosomes. The Mn-induced trafficking of GPP130 occurred from the Golgi via a Rab-7–dependent pathway and did not require its transit through the plasma membrane or early endosomes. Although the cytoplasmic domain of GPP130 was dispensable for its ability to respond to Mn, its lumenal stem domain was required and it had to be targeted to the cis-Golgi for the Mn response to occur. Remarkably, the stem domain was sufficient to confer Mn sensitivity to another cis-Golgi protein. Our results identify the stem domain of GPP130 as a novel Mn sensor in the Golgi lumen of mammalian cells.

The Golgi apparatus plays a central role in processing and sorting proteins and lipids in eukaryotic cells. Golgi compartments constantly exchange material with each other and with other cellular components, allowing them to maintain and reform distinct identities despite dramatic changes in structure and size during cell division, development, and osmotic stress. We have developed three minimal models of membrane and protein exchange in the Golgi\char22{}a discrete, stochastic model, a continuous ordinary differential equation model, and a continuous stochastic differential equation model\char22{}each based on two fundamental mechanisms: vesicle-coat-mediated selective concentration of cargoes and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins during vesicle formation and SNARE-mediated selective fusion of vesicles. By exploring where the models differ, we hope to discover whether the discrete, stochastic nature of vesicle-mediated transport is likely to have appreciable functional consequences for the Golgi. All three models show similar ability to restore and maintain distinct identities over broad parameter ranges. They diverge, however, in conditions corresponding to collapse and reassembly of the Golgi. The results suggest that a continuum model provides a good description of Golgi maintenance but that considering the discrete nature of vesicle-based traffic is important to understanding assembly and disassembly of the Golgi. Experimental analysis validates a prediction of the models that altering guanine nucleotide exchange factor expression levels will modulate Golgi size.

Formation of the ribbon-like membrane network of the Golgi apparatus depends on GM130 and GRASP65, but the mechanism is unknown. We developed an in vivo organelle tethering assaying in which GRASP65 was targeted to the mitochondrial outer membrane either directly or via binding to GM130. Mitochondria bearing GRASP65 became tethered to one another, and this depended on a GRASP65 PDZ domain that was also required for GRASP65 self-interaction. Point mutation within the predicted binding groove of the GRASP65 PDZ domain blocked both tethering and, in a gene replacement assay, Golgi ribbon formation. Tethering also required proximate membrane anchoring of the PDZ domain, suggesting a mechanism that orientates the PDZ binding groove to favor interactions in trans. Thus, a homotypic PDZ interaction mediates organelle tethering in living cells.

Peri-centrosomal positioning of the mammalian Golgi apparatus is known to involve microtubule-based motility, but its importance for cellular physiology is a major unanswered question. Here, we identify golgin-160 and GMAP210 as proteins required for centripetal motility of Golgi membranes. In the absence of either golgin, peri-centrosomal positioning of the Golgi apparatus was disrupted while the cytoskeleton remained intact. Although secretion persisted with normal kinetics, it was evenly distributed in response to wounding rather than directed to the wound edge. Strikingly, these cells also completely failed to polarize. Further, directionally persistent cell migration was inhibited such that wound closure was impaired. These findings not only reveal novel roles for golgin-160 and GMAP210 in conferring membrane motility but also indicate that Golgi positioning has an active role in directed secretion, cell polarity, and wound healing.

The Golgi apparatus is essential for protein sorting and transport. Many researchers have long been fascinated with the form and function of this organelle. Yet, despite decades of scrutiny, the mechanisms by which proteins are transported across the Golgi remain controversial. At a recent meeting, many prominent Golgi researchers assembled to critically evaluate the core issues in the field. This report presents the outcome of their discussions and highlights the key open questions that will help guide the field into a new era.

The synaptic vesicle has been the focus of recent efforts to generate a molecular description of the nerve terminal. The function and molecular composition of the synaptic vesicle are well characterized, due in part to its relative simplicity. Recent work identifies specific proteins involved in some of the functions of the synaptic vesicle and their mechanism of action.