Direct continuities between cisternae at different levels of the Golgi complex in glucose-stimulated mouse islet beta cells

Direct continuity between the membranes of cisternae in the Golgi complex in mammalian cells rarely has been observed; when seen, its documentation has been equivocal. Here we have used dual-axis electron microscope tomography to examine the architecture of the Golgi in three dimensions at approximate to6-nm resolution in rapidly frozen, freeze-substituted murine cells that make and secrete insulin in response to glucose challenge. Our data show three types of direct connections between Golgi cisternae that are normally distinct from one another. These connections all bypass interceding cisternae. We propose that when pancreatic beta cells are stimulated to synthesize and secrete insulin rapidly in vivo, such connections provide a continuous lumen that facilitates the rapid transit of large amounts of newly made protein for secretion. The heterotypic fusion of cisternae, even transiently, raises important questions about the molecular mechanisms that (i) facilitate the fusion/fission of cisternal membranes and control the directionality and specificity of such events, and (it) retain Golgi processing enzymes at specific places within individual cisternae when two cisternae at different levels in the Golgi have fused, maintaining the sequential processing hierarchy that is a hallmark of Golgi organization.

Predicting function from structure: 3D structure studies of the mammalian golgi complex

3D electron tomography studies of the structure of the mammalian Golgi complex have led to four functional predictions (1). The sorting and exit site from the Golgi comprises two or three distinct trans-cisternae (2). The docking of vesicular-tubular clusters at the cis-face and the fragmentation of trans-cisternae are coordinated (3). The mechanisms of transport through, and exit from, the Golgi vary with physiological state, and in different cells and tissues (4). Specialized trans-ER functions in the delivery of ceramide to sphingomyelin synthase in the trans-Golgi membrane, for the regulated sorting via sphingolipid-cholesterol-rich domains. These structure-based predictions can now be tested using a variety of powerful cell and molecular tools.

Cholesterol and fatty acids regulate dynamic caveolin trafficking through the golgi complex and between the cell surface and lipid bodies

Caveolins are a crucial component of plasma membrane (PM) caveolae but have also been localized to intracellular compartments, including the Golgi complex and lipid bodies. Mutant caveolins associated with human disease show aberrant trafficking to the PM and Golgi accumulation. We now show that the Golgi pool of mainly newly synthesized protein is detergent-soluble and predominantly in a monomeric state, in contrast to the surface pool. Caveolin at the PM is not recognized by specific caveolin antibodies unless PM cholesterol is depleted. Exit from the Golgi complex of wild-type caveolin-1 or -3, but not vesicular stomatitis virus-G protein, is modulated by changing cellular cholesterol levels. In contrast, a muscular dystrophy-associated mutant of caveolin-3, Cav3P104L, showed increased accumulation in the Golgi complex upon cholesterol treatment. In addition, we demonstrate that in response to fatty acid treatment caveolin can follow a previously undescribed pathway from the PM to lipid bodies and can move from lipid bodies to the PM in response to removal of fatty acids. The results suggest that cholesterol is a rate-limiting component for caveolin trafficking. Changes in caveolin flux through the exocytic pathway can therefore be an indicator of cellular cholesterol and fatty acid levels.

N4WBP5, a potential target for ubiquitination by the Nedd4 family of proteins, is a novel golgi-associated protein

Nedd4 belongs to a family of ubiquitin-protein ligases that is characterized by 2-4 WW domains, a carboxyl-terminal Hect ((h) under bar omologous to (E) under bar6-AP (C) under bar arboxyl (t) under bar erminus)-domain and in most cases an amino-terminal C2 domain. We had previously identified a series of proteins that associates with the WW domains of Nedd4. In this paper, we demonstrate that one of the Nedd4-binding proteins, N4WBP5, belongs to a small group of evolutionarily conserved proteins with three transmembrane domains. N4WBP5 binds Nedd4 WW domains via the two PPXY motifs present in the amino terminus of the protein. In addition to Nedd4, N4WBP5 can interact with the WW domains of a number of Nedd4 family members and is ubiquitinated. Endogenous N4WBP5 localizes to the Golgi complex. Ectopic expression of the protein disrupts the structure of the Golgi, suggesting that N4WBP5 forms part of a family of integral Golgi membrane proteins. Based on previous observations in yeast, we propose that N4WBP5 may act as an adaptor for Nedd4-like proteins and their putative targets to control ubiquitin-dependent protein sorting and trafficking.

GAIP participates in budding of membrane carriers at the trans-Golgi network

Galpha interacting protein (GAIP) is a regulator of G protein signaling protein that associates dynamically with vesicles and has been implicated in membrane trafficking, although its specific role is not yet known. Using an in vitro budding assay, we show that GAIP is recruited to a specific population of trans-Golgi network-derived vesicles and that these are distinct from coatomer or clathrin-coated vesicles. A truncation mutant (NT-GAIP) encoding only the N-terminal half of GAIP is recruited to trans -Golgi network membranes during the formation of vesicle carriers. Overexpression of NT-GAIP induces the formation of long, coated tubules, which are stabilized by microtubules. Results from the budding assay and from imaging in live cells show that these tubules remain attached to the Golgi stack rather than being released as carrier vesicles. NT-GAIP expression blocks membrane budding and results in the accumulation of tubular carrier intermediates. NT-GAIP-decorated tubules are competent to load vesicular stomatitis virus protein G-green fluorescent protein as post-Golgi, exocytic cargo and in cells expressing NT-GAIP there is reduced surface delivery of vesicular stomatitis virus protein G-green fluorescent protein. We conclude that GAIP functions as an essential part of the membrane budding machinery for a subset of post-Golgi exocytic carriers derived from the trans-Golgi network.

Gp74 a membrane glycoprotein of the cis-Golgi network that cycles through the endoplasmic reticulum and intermediate compartment

A monoclonal antibody CC92 (IgM), raised against a fraction of rat liver enriched in Golgi membranes, recognizes a novel Endo H-resistant 74-kD membrane glycoprotein (gp74). The bulk of gp74 is confined to the cis-Golgi network (CGN). Outside the Golgi gp74 is found in tubulovesicular structures and ER foci. In cells incubated at 37 degrees C the majority of gp74 is segregated from the intermediate compartment (IC) marker p58. However, in cells treated with organelle perturbants such as low temperature, BFA, and [AIF4]- the patterns of the two proteins become indistinguishable. Both proteins are retained in the Golgi complex at 20 degrees C and in the IC at 15 degrees C. Incubation of cells with BFA results in relocation of gp74 to p58 positive IC elements. [AIF4]- induces the redistribution of gp74 from the Golgi to p58-positive vesicles and does not retard the translocation of gp74 to IC elements in cells treated with BFA. Disruption of microtubules by nocodazol results in the rapid disappearance of the Golgi elements stained by gp74 and redistribution of the protein into vesicle-like structures. The responses of gp74 to cell perturbants are in sharp contrast with those of cis/middle and trans-Golgi resident proteins whose location is not affected by low temperatures or [AIF4]-, are translocated to the ER upon addition of BFA, and stay in slow disintegrating Golgi elements in cells treated with nocodazol. The results suggest that gp74 is an itinerant protein that resides most of the time in the CGN and cycles through the ER/IC following the pathway used by p58.

Myosin motors and not actin comets are mediators of the actin-based Golgi-to-endoplasmic reticulum protein transport

We have previously reported that actin filaments are involved in protein transport from the Golgi complex to the endoplasmic reticulum. Herein, we examined whether myosin motors or actin comets mediate this transport. To address this issue we have used, on one hand, a combination of specific inhibitors such as 2,3-butanedione monoxime (BDM) and 1-[5-isoquinoline sulfonyl]-2-methyl piperazine (ML7), which inhibit myosin and the phosphorylation of myosin II by the myosin light chain kinase, respectively; and a mutant of the nonmuscle myosin II regulatory light chain, which cannot be phosphorylated (MRLC2AA). On the other hand, actin comet tails were induced by the overexpression of phosphatidylinositol phosphate 5-kinase. Cells treated with BDM/ML7 or those that express the MRLC2AA mutant revealed a significant reduction in the brefeldin A (BFA)-induced fusion of Golgi enzymes with the endoplasmic reticulum (ER). This delay was not caused by an alteration in the formation of the BFA-induced tubules from the Golgi complex. In addition, the Shiga toxin fragment B transport from the Golgi complex to the ER was also altered. This impairment in the retrograde protein transport was not due to depletion of intracellular calcium stores or to the activation of Rho kinase. Neither the reassembly of the Golgi complex after BFA removal nor VSV-G transport from ER to the Golgi was altered in cells treated with BDM/ML7 or expressing MRLC2AA. Finally, transport carriers containing Shiga toxin did not move into the cytosol at the tips of comet tails of polymerizing actin. Collectively, the results indicate that 1) myosin motors move to transport carriers from the Golgi complex to the ER along actin filaments; 2) nonmuscle myosin II mediates in this process; and 3) actin comets are not involved in retrograde transport.

Kinesin-5/Eg5 is important for transport of CARTS from the trans-Golgi network to the cell surface

Here we report that the kinesin-5 motor Klp61F, which is known for its role in bipolar spindle formation in mitosis, is required for protein transport from the Golgi complex to the cell surface in Drosophila S2 cells. Disrupting the function of its mammalian orthologue, Eg5, in HeLa cells inhibited secretion of a protein called pancreatic adenocarcinoma up-regulated factor (PAUF) but, surprisingly, not the trafficking of vesicular stomatitis virus G protein (VSV-G) to the cell surface. We have previously reported that PAUF is transported from the trans-Golgi network (TGN) to the cell surface in specific carriers called CARTS that exclude VSV-G. Inhibition of Eg5 function did not affect the biogenesis of CARTS; however, their migration was delayed and they accumulated near the Golgi complex. Altogether, our findings reveal a surprising new role of Eg5 in nonmitotic cells in the facilitation of the transport of specific carriers, CARTS, from the TGN to the cell surface.

Apiconuclear Organization of Microtubules Does Not Specify Protein Delivery from the Trans-Golgi Network to Different Membrane Domains in Polarized Epithelial Cells

In nonpolarized epithelial cells, microtubules originate from a broad perinuclear region coincident with the distribution of the Golgi complex and extend outward to the cell periphery (perinuclear [PN] organization). During development of epithelial cell polarity, microtubules reorganize to form long cortical filaments parallel to the lateral membrane, a meshwork of randomly oriented short filaments beneath the apical membrane, and short filaments at the base of the cell; the Golgi becomes localized above the nucleus in the subapical membrane cytoplasm (apiconuclear [AN] organization). The AN-type organization of microtubules is thought to be specialized in polarized epithelial cells to facilitate vesicle trafficking between the trans-Golgi Network (TGN) and the plasma membrane. We describe two clones of MDCK cells, which have different microtubule distributions: clone II/G cells, which gradually reorganize a PN-type distribution of microtubules and the Golgi complex to an AN-type during development of polarity, and clone II/J cells which maintain a PN-type organization. Both cell clones, however, exhibit identical steady-state polarity of apical and basolateral proteins. During development of cell surface polarity, both clones rapidly establish direct targeting pathways for newly synthesized gp80 and gp135/170, and E-cadherin between the TGN and apical and basolateral membrane, respectively; this occurs before development of the AN-type microtubule/Golgi organization in clone II/G cells. Exposure of both clone II/G and II/J cells to low temperature and nocodazole disrupts >99% of microtubules, resulting in: 1) 25–50% decrease in delivery of newly synthesized gp135/170 and E-cadherin to the apical and basolateral membrane, respectively, in both clone II/G and II/J cells, but with little or no missorting to the opposite membrane domain during all stages of polarity development; 2) ∼40% decrease in delivery of newly synthesized gp80 to the apical membrane with significant missorting to the basolateral membrane in newly established cultures of clone II/G and II/J cells; and 3) variable and nonspecific delivery of newly synthesized gp80 to both membrane domains in fully polarized cultures. These results define several classes of proteins that differ in their dependence on intact microtubules for efficient and specific targeting between the Golgi and plasma membrane domains.

A Role for the Lumenal Domain in Golgi Localization of the Saccharomyces cerevisiae Guanosine Diphosphatase

Integral membrane proteins (IMPs) contain localization signals necessary for targeting to their resident subcellular compartments. To define signals that mediate localization to the Golgi complex, we have analyzed a resident IMP of the Saccharomyces cerevisiae Golgi complex, guanosine diphosphatase (GDPase). GDPase, which is necessary for Golgi-specific glycosylation reactions, is a type II IMP with a short amino-terminal cytoplasmic domain, a single transmembrane domain (TMD), and a large catalytic lumenal domain. Regions specifying Golgi localization were identified by analyzing recombinant proteins either lacking GDPase domains or containing corresponding domains from type II vacuolar IMPs. Neither deletion nor substitution of the GDPase cytoplasmic domain perturbed Golgi localization. Exchanging the GDPase TMD with vacuolar protein TMDs only marginally affected Golgi localization. Replacement of the lumenal domain resulted in mislocalization of the chimeric protein from the Golgi to the vacuole, but a similar substitution leaving 34 amino acids of the GDPase lumenal domain intact was properly localized. These results identify a major Golgi localization determinant in the membrane-adjacent lumenal region (stem) of GDPase. Although necessary, the stem domain is not sufficient to mediate localization; in addition, a membrane-anchoring domain and either the cytoplasmic or full-length lumenal domain must be present to maintain Golgi residence. The importance of lumenal domain sequences in GDPase Golgi localization and the requirement for multiple hydrophilic protein domains support a model for Golgi localization invoking protein–protein interactions rather than interactions between the TMD and the lipid bilayer.

A Rab2 Mutant with Impaired GTPase Activity Stimulates Vesicle Formation from Pre-Golgi Intermediates

Rab2 immunolocalizes to pre-Golgi intermediates (vesicular-tubular clusters [VTCs]) that are the first site of segregation of anterograde- and retrograde-transported proteins and a major peripheral site for COPI recruitment. Our previous work showed that Rab2 Q65L (equivalent to Ras Q61L) inhibited endoplasmic reticulum (ER)-to-Golgi transport in vivo. In this study, the biochemical properties of Rab2 Q65L were analyzed. The mutant protein binds GDP and GTP and has a low GTP hydrolysis rate that suggests that Rab2 Q65L is predominantly in the GTP-bound–activated form. The purified protein arrests vesicular stomatitis virus glycoprotein transport from VTCs in an assay that reconstitutes ER-to-Golgi traffic. A quantitative binding assay was used to measure membrane binding of β-COP when incubated with the mutant. Unlike Rab2 that stimulates recruitment, Rab2 Q65L showed a dose-dependent decrease in membrane-associated β-COP when incubated with rapidly sedimenting membranes (ER, pre-Golgi, and Golgi). The mutant protein does not interfere with β-COP binding but stimulates the release of slowly sedimenting vesicles containing Rab2, β-COP, and p53/gp58 but lacking anterograde grade-directed cargo. To complement the biochemical results, we observed in a morphological assay that Rab2 Q65L caused vesiculation of VTCs that accumulated at 15°C. These data suggest that the Rab2 protein plays a role in the low-temperature–sensitive step that regulates membrane flow from VTCs to the Golgi complex and back to the ER.

Vesicle-associated Membrane Protein 4 is Implicated in Trans-Golgi Network Vesicle Trafficking

The trans-Golgi network (TGN) plays a pivotal role in directing proteins in the secretory pathway to the appropriate cellular destination. VAMP4, a recently discovered member of the vesicle-associated membrane protein (VAMP) family of trafficking proteins, has been suggested to play a role in mediating TGN trafficking. To better understand the function of VAMP4, we examined its precise subcellular distribution. Indirect immunofluorescence and electron microscopy revealed that the majority of VAMP4 localized to tubular and vesicular membranes of the TGN, which were in part coated with clathrin. In these compartments, VAMP4 was found to colocalize with the putative TGN-trafficking protein syntaxin 6. Additional labeling was also present on clathrin-coated and noncoated vesicles, on endosomes and the medial and trans side of the Golgi complex, as well as on immature secretory granules in PC12 cells. Immunoprecipitation of VAMP4 from rat brain detergent extracts revealed that VAMP4 exists in a complex containing syntaxin 6. Converging lines of evidence implicate a role for VAMP4 in TGN-to-endosome transport.

Mapmodulin, Cytoplasmic Dynein, and Microtubules Enhance the Transport of Mannose 6-Phosphate Receptors from Endosomes to the Trans-Golgi Network

Late endosomes and the Golgi complex maintain their cellular localizations by virtue of interactions with the microtubule-based cytoskeleton. We study the transport of mannose 6-phosphate receptors from late endosomes to the trans-Golgi network in vitro. We show here that this process is facilitated by microtubules and the microtubule-based motor cytoplasmic dynein; transport is inhibited by excess recombinant dynamitin or purified microtubule-associated proteins. Mapmodulin, a protein that interacts with the microtubule-associated proteins MAP2, MAP4, and tau, stimulates the microtubule- and dynein-dependent localization of Golgi complexes in semi-intact Chinese hamster ovary cells. The present study shows that mapmodulin also stimulates the initial rate with which mannose 6-phosphate receptors are transported from late endosomes to the trans-Golgi network in vitro. These findings represent the first indication that mapmodulin can stimulate a vesicle transport process, and they support a model in which the microtubule-based cytoskeleton enhances the efficiency of vesicle transport between membrane-bound compartments in mammalian cells.

The Dynamics of Golgi Protein Traffic Visualized in Living Yeast Cells

We describe for the first time the visualization of Golgi membranes in living yeast cells, using green fluorescent protein (GFP) chimeras. Late and early Golgi markers are present in distinct sets of scattered, moving cisternae. The immediate effects of temperature-sensitive mutations on the distribution of these markers give clues to the transport processes occurring. We show that the late Golgi marker GFP-Sft2p and the glycosyltransferases, Anp1p and Mnn1p, disperse into vesicle-like structures within minutes of a temperature shift in sec18, sft1, and sed5 cells, but not in sec14 cells. This is consistent with retrograde vesicular traffic, mediated by the vesicle SNARE Sft1p, to early cisternae containing the target SNARE Sed5p. Strikingly, Sed5p itself moves rapidly to the endoplasmic reticulum (ER) in sec12 cells, implying that it cycles through the ER. Electron microscopy shows that Golgi membranes vesiculate in sec18 cells within 10 min of a temperature shift. These results emphasize the dynamic nature of Golgi cisternae and satisfy the kinetic requirements of a cisternal maturation model in which all resident proteins must undergo retrograde vesicular transport, either within the Golgi complex or from there to the ER, as anterograde cargo advances.

Cholesterol-independent Targeting of Golgi Membrane Proteins in Insect Cells

Distinct lipid compositions of intracellular organelles could provide a physical basis for targeting of membrane proteins, particularly where transmembrane domains have been shown to play a role. We tested the possibility that cholesterol is required for targeting of membrane proteins to the Golgi complex. We used insect cells for our studies because they are cholesterol auxotrophs and can be depleted of cholesterol by growth in delipidated serum. We found that two well-characterized mammalian Golgi proteins were targeted to the Golgi region of Aedes albopictus cells, both in the presence and absence of cellular cholesterol. Our results imply that a cholesterol gradient through the secretory pathway is not required for membrane protein targeting to the Golgi complex, at least in insect cells.