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1.  Manganese induces oligomerization to promote down-regulation of the intracellular trafficking receptor used by Shiga toxin 
Molecular Biology of the Cell  2014;25(19):3049-3058.
Manganese down-regulates the Shiga toxin receptor GPP130, which protects against lethal toxin doses. This study reveals a major aspect of the mechanism. Manganese binds GPP130, inducing GPP130 oligomerization, which is required and sufficient to redirect GPP130 out of the Golgi toward lysosomes.
Manganese (Mn) protects cells against lethal doses of purified Shiga toxin by causing the degradation of the cycling transmembrane protein GPP130, which the toxin uses as a trafficking receptor. Mn-induced GPP130 down-regulation, in addition to being a potential therapeutic approach against Shiga toxicosis, is a model for the study of metal-regulated protein sorting. Significantly, however, the mechanism by which Mn regulates GPP130 trafficking is unknown. Here we show that a transferable trafficking determinant within GPP130 bound Mn and that Mn binding induced GPP130 oligomerization in the Golgi. Alanine substitutions blocking Mn binding abrogated both oligomerization of GPP130 and GPP130 sorting from the Golgi to lysosomes. Further, oligomerization was sufficient because forced aggregation, using a drug-controlled polymerization domain, redirected GPP130 to lysosomes in the absence of Mn. These experiments reveal metal-induced oligomerization as a Golgi sorting mechanism for a medically relevant receptor for Shiga toxin.
doi:10.1091/mbc.E14-05-1003
PMCID: PMC4230593  PMID: 25079690
2.  Retrograde trafficking of AB5 toxins: mechanisms to therapeutics 
Bacterial AB5 toxins are a clinically relevant class of exotoxins that includes 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.
doi:10.1007/s00109-013-1048-7
PMCID: PMC3773303  PMID: 23665994
3.  GRASP65 controls the cis Golgi integrity in vivo 
Biology Open  2014;3(6):431-443.
ABSTRACT
GRASP65 and GRASP55 are peripheral Golgi proteins localized to cis and medial/trans cisternae, respectively. They are implicated in diverse aspects of protein transport and structure related to the Golgi complex, including the stacking of the Golgi stack and/or the linking of mammalian Golgi stacks into the Golgi ribbon. Using a mouse model, we interfered with GRASP65 by homologous recombination and confirmed its absence of expression. Surprisingly, the mice were healthy and fertile with no apparent defects in tissue, cellular or subcellular organization. Immortalized MEFs derived from the mice did not show any growth or morphological defects. However, despite the normal appearance of the Golgi ribbon, a fluorescence recovery after photobleaching assay revealed functional discontinuities specific to the cis cisternal membrane network. This leads to a strong change in the plasma membrane GSII lectin staining that was also observed in certain mutant tissues. These findings substantiate the role of GRASP65 in continuity of the cis Golgi network required for proper glycosylation, while showing that neither this continuity nor GRASP65 itself are essential for the viability of a complex organism.
doi:10.1242/bio.20147757
PMCID: PMC4058077  PMID: 24795147
Knock-in mouse; GRASP65; GORASP; GRASP55; FRAP; Golgi; Glycosylation; Expression pattern; Golgi ribbon linking
4.  Isoform-specific tethering links the Golgi ribbon to maintain compartmentalization 
Molecular Biology of the Cell  2014;25(1):133-144.
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.
doi:10.1091/mbc.E13-07-0395
PMCID: PMC3873884  PMID: 24227884
5.  Binding of the vesicle docking protein p115 to the GTPase Rab1b regulates membrane recruitment of the COPI vesicle coat 
Cellular Logistics  2014;3:e27687.
Membrane recruitment of the COPI vesicle coat is fundamental to its function and contributes to compartment identity in the early secretory pathway. COPI recruitment is triggered by guanine nucleotide exchange activating the Arf1 GTPase, but the key exchange factor, GBF1, is a peripheral membrane component whose membrane association is dependent on another GTPase, Rab1. Inactive Rab GTPases are in a soluble complex with guanine nucleotide dissociation inhibitor (GDI) and activation of Rab GTPases by exchange factors can be enhanced by GDI dissociation factors (GDFs). In the present study, we investigated the vesicle docking protein p115 and it’s binding to the Rab1 isoform Rab1b. Inhibition of p115 expression induced dissociation of Rab1b from Golgi membranes. Rab1b bound the cc2 domain of p115 and p115 lacking this domain failed to recruit Rab1b. Further, p115 inhibition blocked association of the COPI coat with Golgi membranes and this was suppressed by constitutive activation of Rab1b. These findings show p115 enhancement of Rab1b activation leading to COPI recruitment suggesting a connection between the vesicle docking machinery and the vesicle coat complex during the establishment of post-ER compartment identity.
doi:10.4161/cl.27687
PMCID: PMC4187009  PMID: 25332841
vesicle tether; vesicle coat complex; Rab GTPase; p115; COPI
6.  A Sensor of Protein O-Glycosylation Based on Sequential Processing in the Golgi apparatus 
Traffic (Copenhagen, Denmark)  2012;14(1):47-56.
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 sum, 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.
doi:10.1111/tra.12019
PMCID: PMC3548014  PMID: 23046148
O-glycosylation; fluorescence; biosensor; Golgi complex; furin
7.  Shiga toxin–binding site for host cell receptor GPP130 reveals unexpected divergence in toxin-trafficking mechanisms 
Molecular Biology of the Cell  2013;24(15):2311-2318.
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.
doi:10.1091/mbc.E13-01-0057
PMCID: PMC3727924  PMID: 23761068
8.  Golgin160 Recruits the Dynein Motor to Position the Golgi Apparatus 
Developmental cell  2012;23(1):153-165.
Membrane motility is a fundamental characteristic of all eukaryotic cells. One of the best-known examples is that of the mammalian Golgi apparatus, where constant inward movement of Golgi membranes results in its characteristic position near the centrosome. While it is clear that the minus-end-directed motor dynein is required for this process, the mechanism and regulation of dynein recruitment to Golgi membranes remains unknown. Here, we show that the Golgi protein golgin160 recruits dynein to Golgi membranes. This recruitment confers centripetal motility to membranes, and is regulated by the GTPase Arf1. Further, during cell division, motor association with membranes is regulated by the dissociation of the receptor-motor complex from membranes. These results identify a cell cycle-regulated membrane receptor for a molecular motor, and suggest a mechanistic basis for achieving the dramatic changes in organelle positioning seen during cell division.
doi:10.1016/j.devcel.2012.05.023
PMCID: PMC3417773  PMID: 22814606
9.  Golgi Positioning 
Movement of dynein motor proteins along microtubules dynamically positions the Golgi apparatus close to centrosomes. Proteins such as lava lamp, golgin-160, or GMAP210 may help link these motors to Golgi membranes.
The Golgi apparatus in mammalian cells is positioned near the centrosome-based microtubule-organizing center (Fig. 1). Secretory cargo moves inward in membrane carriers for delivery to Golgi membranes in which it is processed and packaged for transport outward to the plasma membrane. Cytoplasmic dynein motor proteins (herein termed dynein) primarily mediate inward cargo carrier movement and Golgi positioning. These motors move along microtubules toward microtubule minus-ends embedded in centrosomes. Centripetal motility is controlled by a host of regulators whose precise functions remain to be determined. Significantly, a specific Golgi receptor for dynein has not been identified. This has impaired progress toward elucidation of membrane-motor-microtubule attachment in the periphery and, after inward movement, recycling of the motor for another round. Pericentrosomal positioning of the Golgi apparatus is dynamic. It is regulated during critical cellular processes such as mitosis, differentiation, cell polarization, and cell migration. Positioning is also important as it aligns the Golgi along an axis of cell polarity. In certain cell types, this promotes secretion directed to the proximal plasma membrane domain thereby maintaining specializations critical for diverse processes including wound healing, immunological synapse formation, and axon determination.
doi:10.1101/cshperspect.a005322
PMCID: PMC3101843  PMID: 21504874
10.  Active Mask Segmentation of Fluorescence Microscope Images 
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 “contour” to that of “inside and outside”, 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.
doi:10.1109/TIP.2009.2021081
PMCID: PMC2765110  PMID: 19380268
11.  Manganese-induced Trafficking and Turnover of the cis-Golgi Glycoprotein GPP130 
Molecular Biology of the Cell  2010;21(7):1282-1292.
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.
doi:10.1091/mbc.E09-11-0985
PMCID: PMC2847531  PMID: 20130081
12.  Journeys through the Golgi—taking stock in a new era 
The Journal of Cell Biology  2009;187(4):449-453.
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.
doi:10.1083/jcb.200909011
PMCID: PMC2779233  PMID: 19948493
13.  Organelle tethering by a homotypic PDZ interaction underlies formation of the Golgi membrane network 
The Journal of Cell Biology  2009;186(1):41-55.
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.
doi:10.1083/jcb.200902110
PMCID: PMC2712994  PMID: 19581411
14.  A Primary Role for Golgi Positioning in Directed Secretion, Cell Polarity, and Wound Healing 
Molecular Biology of the Cell  2009;20(6):1728-1736.
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.
doi:10.1091/mbc.E08-10-1077
PMCID: PMC2655245  PMID: 19158377
15.  Coat-Tether Interaction in Golgi Organization 
Molecular Biology of the Cell  2008;19(7):2830-2843.
Biogenesis of the Golgi apparatus is likely mediated by the COPI vesicle coat complex, but the mechanism is poorly understood. Modeling of the COPI subunit βCOP based on the clathrin adaptor AP2 suggested that the βCOP C terminus forms an appendage domain with a conserved FW binding pocket motif. On gene replacement after knockdown, versions of βCOP with a mutated FW motif or flanking basic residues yielded a defect in Golgi organization reminiscent of that occurring in the absence of the vesicle tether p115. Indeed, βCOP bound p115, and this depended on the βCOP FW motif. Furthermore, the interaction depended on E19E21 in the p115 head domain and inverse charge substitution blocked Golgi biogenesis in intact cells. Finally, Golgi assembly in permeabilized cells was significantly reduced by inhibitors containing intact, but not mutated, βCOP FW or p115 EE motifs. Thus, Golgi organization depends on mutually interacting domains in βCOP and p115, suggesting that vesicle tethering at the Golgi involves p115 binding to the COPI coat.
doi:10.1091/mbc.E07-12-1236
PMCID: PMC2441675  PMID: 18434597
16.  GRASP55 Regulates Golgi Ribbon Formation 
Molecular Biology of the Cell  2008;19(7):2696-2707.
Recent work indicates that mitogen-activated protein kinase kinase (MEK)1 signaling at the G2/M cell cycle transition unlinks the contiguous mammalian Golgi apparatus and that this regulates cell cycle progression. Here, we sought to determine the role in this pathway of Golgi reassembly protein (GRASP)55, a Golgi-localized target of MEK/extracellular signal-regulated kinase (ERK) phosphorylation at mitosis. In support of the hypothesis that GRASP55 is inhibited in late G2 phase, causing unlinking of the Golgi ribbon, we found that HeLa cells depleted of GRASP55 show a fragmented Golgi similar to control cells arrested in G2 phase. In the absence of GRASP55, Golgi stack length is shortened but Golgi stacking, compartmentalization, and transport seem normal. Absence of GRASP55 was also sufficient to suppress the requirement for MEK1 in the G2/M transition, a requirement that we previously found depends on an intact Golgi ribbon. Furthermore, mimicking mitotic phosphorylation of GRASP55 by using aspartic acid substitutions is sufficient to unlink the Golgi apparatus in a gene replacement assay. Our results implicate MEK1/ERK regulation of GRASP55-mediated Golgi linking as a control point in cell cycle progression.
doi:10.1091/mbc.E07-11-1200
PMCID: PMC2441664  PMID: 18434598
17.  Mitogen-activated Protein Kinase Kinase 1-dependent Golgi Unlinking Occurs in G2 Phase and Promotes the G2/M Cell Cycle Transition 
Molecular Biology of the Cell  2007;18(2):594-604.
Two controversies have emerged regarding the signaling pathways that regulate Golgi disassembly at the G2/M cell cycle transition. The first controversy concerns the role of mitogen-activated protein kinase activator mitogen-activated protein kinase kinase (MEK)1, and the second controversy concerns the participation of Golgi structure in a novel cell cycle “checkpoint.” A potential simultaneous resolution is suggested by the hypothesis that MEK1 triggers Golgi unlinking in late G2 to control G2/M kinetics. Here, we show that inhibition of MEK1 by RNA interference or by using the MEK1/2-specific inhibitor U0126 delayed the passage of synchronized HeLa cells into M phase. The MEK1 requirement for normal mitotic entry was abrogated if Golgi proteins were dispersed before M phase by treatment of cells with brefeldin A or if GRASP65, which links Golgi stacks into a ribbon network, was depleted. Imaging revealed that unlinking of the Golgi apparatus begins before M phase, is independent of cyclin-dependent kinase 1 activation, and requires MEK signaling. Furthermore, expression of the GRASP family member GRASP55 after alanine substitution of its MEK1-dependent mitotic phosphorylation sites inhibited both late G2 Golgi unlinking and the G2/M transition. Thus, MEK1 plays an in vivo role in Golgi reorganization, which regulates cell cycle progression.
doi:10.1091/mbc.E06-06-0530
PMCID: PMC1783781  PMID: 17182854
18.  COPII–Golgi protein interactions regulate COPII coat assembly and Golgi size 
The Journal of Cell Biology  2006;174(1):53-63.
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.
doi:10.1083/jcb.200604058
PMCID: PMC2064162  PMID: 16818719
19.  A Cycling cis-Golgi Protein Mediates Endosome-to-Golgi Traffic 
Molecular Biology of the Cell  2004;15(11):4798-4806.
Toxins can invade cells by using a direct endosome-to-Golgi endocytic pathway that bypasses late endosomes/prelysosomes. This is also a route used by endogenous proteins, including GPP130, which is an integral membrane protein retrieved via the bypass pathway from endosomes to its steady-state location in the cis-Golgi. An RNA interference-based test revealed that GPP130 was required for efficient exit of Shiga toxin B-fragment from endosomes en route to the Golgi apparatus. Furthermore, two proteins whose Golgi targeting depends on endosome-to-Golgi retrieval in the bypass pathway accumulated in early/recycling endosomes in the absence of GPP130. GPP130 activity seemed specific to bypass pathway trafficking because the targeting of other tested proteins, including those retrieved to the Golgi via the more conventional late endosome route, was unaltered. Thus, a distally cycling Golgi protein mediates exit from endosomes and thereby underlies Shiga toxin invasion and retrieval-based targeting of other cycling Golgi proteins.
doi:10.1091/mbc.E04-05-0366
PMCID: PMC524728  PMID: 15331763
20.  Capacity of the Golgi Apparatus for Biogenesis from the Endoplasmic Reticulum 
Molecular Biology of the Cell  2003;14(12):5011-5018.
It is unclear whether the mammalian Golgi apparatus can form de novo from the ER or whether it requires a preassembled Golgi matrix. As a test, we assayed Golgi reassembly after forced redistribution of Golgi matrix proteins into the ER. Two conditions were used. In one, ER redistribution was achieved using a combination of brefeldin A (BFA) to cause Golgi collapse and H89 to block ER export. Unlike brefeldin A alone, which leaves matrix proteins in relatively large remnant structures outside the ER, the addition of H89 to BFA-treated cells caused ER accumulation of all Golgi markers tested. In the other, clofibrate treatment induced ER redistribution of matrix and nonmatrix proteins. Significantly, Golgi reassembly after either treatment was robust, implying that the Golgi has the capacity to form de novo from the ER. Furthermore, matrix proteins reemerged from the ER with faster ER exit rates. This, together with the sensitivity of BFA remnants to ER export blockade, suggests that presence of matrix proteins in BFA remnants is due to cycling via the ER and preferential ER export rather than their stable assembly in a matrix outside the ER. In summary, the Golgi apparatus appears capable of efficient self-assembly.
doi:10.1091/mbc.E03-06-0437
PMCID: PMC284802  PMID: 14565973
21.  Basolateral Cycling Mediated by a Lumenal Domain Targeting Determinant 
Molecular Biology of the Cell  2003;14(5):1801-1807.
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.
doi:10.1091/mbc.E02-10-0692
PMCID: PMC165078  PMID: 12802056
22.  Evidence that Golgi structure depends on a p115 activity that is independent of the vesicle tether components giantin and GM130 
The Journal of Cell Biology  2001;155(2):227-238.
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.
doi:10.1083/jcb.200105005
PMCID: PMC2198842  PMID: 11591729
Golgi structure; tether; docking; mitosis; COPI
23.  Lumenal Endosomal and Golgi-Retrieval Determinants Involved in pH-sensitive Targeting of an Early Golgi Protein 
Molecular Biology of the Cell  2001;12(10):3152-3160.
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 a cis-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.
PMCID: PMC60163  PMID: 11598199
24.  Osmotically Induced Cell Volume Changes Alter Anterograde and Retrograde Transport, Golgi Structure, and COPI Dissociation 
Molecular Biology of the Cell  1999;10(5):1445-1462.
Physiological conditions that impinge on constitutive traffic and affect organelle structure are not known. We report that osmotically induced cell volume changes, which are known to occur under a variety of conditions, rapidly inhibited endoplasmic reticulum (ER)-to-Golgi transport in mammalian cells. Both ER export and ER Golgi intermediate compartment (ERGIC)-to-Golgi trafficking steps were blocked, but retrograde transport was active, and it mediated ERGIC and Golgi collapse into the ER. Extensive tubulation and relatively rapid Golgi resident redistribution were observed under hypo-osmotic conditions, whereas a slower redistribution of the same markers, without apparent tubulation, was observed under hyperosmotic conditions. The osmotic stress response correlated with the perturbation of COPI function, because both hypo- and hyperosmotic conditions slowed brefeldin A-induced dissociation of βCOP from Golgi membranes. Remarkably, Golgi residents reemerged after several hours of sustained incubation in hypotonic or hypertonic medium. Reemergence was independent of new protein synthesis but required PKC, an activity known to mediate cell volume recovery. Taken together these results indicate the existence of a coupling between cell volume and constitutive traffic that impacts organelle structure through independent effects on anterograde and retrograde flow and that involves, in part, modulation of COPI function.
PMCID: PMC25298  PMID: 10233155
25.  The Golgi and Endoplasmic Reticulum Remain Independent during Mitosis in HeLa Cells 
Molecular Biology of the Cell  1998;9(3):623-635.
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.
PMCID: PMC25291  PMID: 9487131

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