PtdIns4P recognition by Vps74/GOLPH3 links PtdIns 4-kinase signaling to retrograde Golgi trafficking

Targeting and retention of resident integral membrane proteins of the Golgi apparatus underly the function of the Golgi in glycoprotein and glycolipid processing and sorting. In yeast, steady-state Golgi localization of multiple mannosyltransferases requires recognition of their cytosolic domains by the peripheral Golgi membrane protein Vps74, an orthologue of human GOLPH3/GPP34/GMx33/MIDAS (mitochondrial DNA absence sensitive factor). We show that targeting of Vps74 and GOLPH3 to the Golgi apparatus requires ongoing synthesis of phosphatidylinositol (PtdIns) 4-phosphate (PtdIns4P) by the Pik1 PtdIns 4-kinase and that modulation of the levels and cellular location of PtdIns4P leads to mislocalization of these proteins. Vps74 and GOLPH3 bind specifically to PtdIns4P, and a sulfate ion in a crystal structure of GOLPH3 indicates a possible phosphoinositide-binding site that is conserved in Vps74. Alterations in this site abolish phosphoinositide binding in vitro and Vps74 function in vivo. These results implicate Pik1 signaling in retention of Golgi-resident proteins via Vps74 and show that GOLPH3 family proteins are effectors of Golgi PtdIns 4-kinases.     

An essential cytoplasmic domain for the Golgi localization of coiled-coil proteins with a COOH-terminal membrane anchor

Giantin is a resident Golgi protein that has an extremely long cytoplasmic domain (about 370 kDa) and is anchored to the Golgi membrane by the COOH-terminal membrane-anchoring domain (CMD) with no luminal extension. We examined the essential domain of giantin required for Golgi localization by mutational analysis. The Golgi localization of giantin was not affected by the deletion of its CMD or by substitution with the CMD of syntaxin-2, a plasma membrane protein. The giantin CMD fused to the cytoplasmic domain of syntaxin-2 could not retain the chimera in the Golgi apparatus. Sequential deletion analysis showed that the COOH-terminal sequence (positions 3059--3161) adjacent to the CMD was the essential domain required for the Golgi localization of giantin. We also examined two other Golgi-resident proteins, golgin-84 and syntaxin-5, with a similar membrane topology as giantin. It was confirmed that the cytoplasmic domain of about 100 residues adjacent to the CMD was required for their Golgi localization. Taken together, these results suggest that the COOH-terminally anchored Golgi proteins with long cytoplasmic extensions have the Golgi localization signal(s) in the cytoplasmic sequence adjacent to the CMD. This is in contrast to previous observations that a transmembrane domain is required for Golgi localization by other Golgi proteins transported from the endoplasmic reticulum.     

Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wall biosynthesis

The plant Golgi plays a pivotal role in the biosynthesis of cell wall matrix polysaccharides, protein glycosylation, and vesicle trafficking. Golgi-localized proteins have become prospective targets for reengineering cell wall biosynthetic pathways for the efficient production of biofuels from plant cell walls. However, proteomic characterization of the Golgi has so far been limited, owing to the technical challenges inherent in Golgi purification. In this study, a combination of density centrifugation and surface charge separation techniques have allowed the reproducible isolation of Golgi membranes from Arabidopsis (Arabidopsis thaliana) at sufficiently high purity levels for in-depth proteomic analysis. Quantitative proteomic analysis, immunoblotting, enzyme activity assays, and electron microscopy all confirm high purity levels. A composition analysis indicated that approximately 19% of proteins were likely derived from contaminating compartments and ribosomes. The localization of 13 newly assigned proteins to the Golgi using transient fluorescent markers further validated the proteome. A collection of 371 proteins consistently identified in all replicates has been proposed to represent the Golgi proteome, marking an appreciable advancement in numbers of Golgi-localized proteins. A significant proportion of proteins likely involved in matrix polysaccharide biosynthesis were identified. The potential within this proteome for advances in understanding Golgi processes has been demonstrated by the identification and functional characterization of the first plant Golgi-resident nucleoside diphosphatase, using a yeast complementation assay. Overall, these data show key proteins involved in primary cell wall synthesis and include a mixture of well-characterized and unknown proteins whose biological roles and importance as targets for future research can now be realized.     

Glycosylphosphatidylinositol biosynthetic enzymes are localized to a stable tubular subcompartment of the endoplasmic reticulum in Leishmania mexicana.

Glycosylphosphatidylinositols (GPI) are essential components in the plasma membrane of the protozoan parasite Leishmania mexicana, both as membrane anchors for the major surface macromolecules and as the sole class of free glycolipids. We provide evidence that L.mexicana dolichol-phosphate-mannose synthase (DPMS), a key enzyme in GPI biosynthesis, is localized to a distinct tubular subdomain of the endoplasmic reticulum (ER), based on the localization of a green fluorescent protein (GFP)-DPMS chimera and subcellular fractionation experiments. This tubular membrane (termed the DPMS tubule) is also enriched in other enzymes involved in GPI biosynthesis, can be specifically stained with the fluorescent lipid, BODIPY-C5-ceramide, and appears to be connected to specific subpellicular microtubules that underlie the plasma membrane. Perturbation of microtubules and DPMS tubule structure in vivo results in the selective accumulation of GPI anchor precursors, but not free GPIs. The DPMS tubule is closely associated morphologically with the single Golgi apparatus in non-dividing and dividing cells, appears to exclude luminal ER resident proteins and is labeled, together with the Golgi apparatus, with another GFP chimera containing the heterologous human Golgi marker beta1,2-N-acetylglucosaminyltransferase-I. The possibility that the DPMS-tubule is a stable transitional ER is discussed.     

Yeast Dop1 is required for glycosyltransferase retrieval from the trans-Golgi network

BackgroundGlycosyltransferases are type II membrane proteins that are responsible for glycan modification of proteins and lipids, and localize to distinct cisternae in the Golgi apparatus. During cisternal maturation, retrograde trafficking helps maintain the steady-state localization of these enzymes in the sub-compartments of the Golgi.MethodsTo understand how glycosyltransferases are recycled in the late Golgi complex, we searched for genes that are essential for budding yeast cell growth and that encode proteins localized in endosomes and in the Golgi. We specifically analyzed the roles of Dop1 and its binding partner Neo1 in retaining Golgi-resident glycosyltransferases, in the late Golgi complex.ResultsDop1 primarily localized to younger compartments of the trans-Golgi network (TGN) and seemed to cycle within the TGN. In contrast, Neo1, a P4-ATPase that interacts with Dop1, localized to the TGN. Abolition of DOP1 expression led to defects in the FM4-64 endocytic pathway. Dop1 and Neo1 were required for correct glycosylation of invertase, a secretory protein, at the Golgi. In DOP1-shutdown cells, Och1, a mannosyltransferase that is typically located in the cis-Golgi, mislocalized to the TGN. In addition, the function of multiple glycosyltransferases required for N- and O-glycosylation were impaired in DOP1-shutdown cells.ConclusionsOur results indicate that Dop1 is involved in vesicular transport at the TGN, and is critical for retrieving glycosyltransferases from the TGN to the Golgi in yeast.General significanceGolgi-resident glycosyltransferases recycling from the TGN to the Golgi is dependent on Dop1 and the P4-ATPase Neo1.     

Possible implication of Golgi-nucleating function for the centrosome

The Golgi apparatus breaks down at mitosis, resulting in the dispersal of Golgi-resident proteins. In NRK cells, however, subsets of both TGN38 and golgin-97, but not ManII and GM130, remained associated with the centrosome throughout the cell cycle. This centrosome association of TGN38 and golgin-97 was not disrupted by treatment with brefeldin A, additional inducers of retrograde trafficking and inhibitors of either kinases or protein phosphatases. Anchoring of the Golgi apparatus within the juxtanuclear region depends on microtubules; the association of TGN38 and golgin-97 subsets with the centrosome, however, was insensitive to nocodazole treatment. Drugs such as PDMP, which block Golgi dispersal both by nocodazole, despite microtubule depolymerization, and by inducers of retrograde trafficking, strengthened the microtubule-nucleating activity of the centrosome. These observations cumulatively suggest the centrosome is implicated in nucleation of the Golgi apparatus through interactions with Golgi-resident proteins, such as TGN38 and golgin-97.     

Proteomic identification of S-nitrosylated Golgi proteins: new insights into endothelial cell regulation by eNOS-derived NO

Background

Endothelial nitric oxide synthase (eNOS) is primarily localized on the Golgi apparatus and plasma membrane caveolae in endothelial cells. Previously, we demonstrated that protein S-nitrosylation occurs preferentially where eNOS is localized. Thus, in endothelial cells, Golgi proteins are likely to be targets for S-nitrosylation. The aim of this study was to identify S-nitrosylated Golgi proteins and attribute their S-nitrosylation to eNOS-derived nitric oxide in endothelial cells.

Methods

Golgi membranes were isolated from rat livers. S-nitrosylated Golgi proteins were determined by a modified biotin-switch assay coupled with mass spectrometry that allows the identification of the S-nitrosylated cysteine residue. The biotin switch assay followed by Western blot or immunoprecipitation using an S-nitrosocysteine antibody was also employed to validate S-nitrosylated proteins in endothelial cell lysates.

Results

Seventy-eight potential S-nitrosylated proteins and their target cysteine residues for S-nitrosylation were identified; 9 of them were Golgi-resident or Golgi/endoplasmic reticulum (ER)-associated proteins. Among these 9 proteins, S-nitrosylation of EMMPRIN and Golgi phosphoprotein 3 (GOLPH3) was verified in endothelial cells. Furthermore, S-nitrosylation of these proteins was found at the basal levels and increased in response to eNOS stimulation by the calcium ionophore A23187. Immunofluorescence microscopy and immunoprecipitation showed that EMMPRIN and GOLPH3 are co-localized with eNOS at the Golgi apparatus in endothelial cells. S-nitrosylation of EMMPRIN was notably increased in the aorta of cirrhotic rats.

Conclusion

Our data suggest that the selective S-nitrosylation of EMMPRIN and GOLPH3 at the Golgi apparatus in endothelial cells results from the physical proximity to eNOS-derived nitric oxide.     

Endogenous tagging reveals a mid-Golgi localization of the glycosyltransferase-cleaving intramembrane protease SPPL3

Numerous Golgi-resident enzymes implicated in glycosylation are regulated by the conserved intramembrane protease SPPL3. SPPL3-catalyzed endoproteolysis separates Golgi enzymes from their membrane anchors, enabling subsequent release from the Golgi and secretion. Experimentally altered SPPL3 expression changes glycosylation patterns, yet the regulation of SPPL3-mediated Golgi enzyme cleavage is not understood and conflicting results regarding the subcellular localization of SPPL3 have been reported. Here, we used precise genome editing to generate isogenic cell lines expressing N- or C-terminally tagged SPPL3 from its endogenous locus. Using these cells, we conducted co-localization analyses of tagged endogenous SPPL3 and Golgi markers under steady-state conditions and upon treatment with drugs disrupting Golgi organization. Our data demonstrate that endogenous SPPL3 is Golgi-resident and found predominantly in the mid-Golgi. We find that endogenous SPPL3 co-localizes with its substrates but similarly with non-substrate type II proteins, demonstrating that in addition to co-localization in the Golgi other substrate-intrinsic properties govern SPPL3-mediated intramembrane proteolysis. Given the prevalence of SPPL3-mediated cleavage among Golgi-resident proteins our results have important implications for the regulation of SPPL3 and its role in the organization of the Golgi glycosylation machinery.     

Golgi-Resident GTPase Rab30 Promotes the Biogenesis of Pathogen-Containing Autophagosomes

Autophagy acts as a host-defense system against pathogenic microorganisms such as Group A Streptococcus (GAS). Autophagy is a membrane-mediated degradation system that is regulated by intracellular membrane trafficking regulators, including small GTPase Rab proteins. Here, we identified Rab30 as a novel regulator of GAS-containing autophagosome-like vacuoles (GcAVs). We found that Rab30, a Golgi-resident Rab, was recruited to GcAVs in response to autophagy induction by GAS infection in epithelial cells. Rab30 recruitment was dependent upon its GTPase activity. In addition, the knockdown of Rab30 expression significantly reduced GcAV formation efficiency and impaired intracellular GAS degradation. Rab30 normally functions to maintain the structural integrity of the Golgi complex, but GcAV formation occurred even when the Golgi apparatus was disrupted. Although Rab30 also colocalized with a starvation-induced autophagosome, Rab30 was not required for autophagosome formation during starvation. These results suggest that Rab30 mediates autophagy against GAS independently of its normal cellular role in the structural maintenance of the Golgi apparatus, and autophagosome biogenesis during bacterial infection involves specific Rab GTPases.     

Maintenance of the diacylglycerol level in the Golgi apparatus by the Nir2 protein is critical for Golgi secretory function

The level of diacylglycerol (DAG) in the Golgi apparatus is crucial for protein transport to the plasma membrane. Studies in budding yeast indicate that Sec14p, a phosphatidylinositol (PI)-transfer protein, is involved in regulating DAG homeostasis in the Golgi complex. Here, we show that Nir2, a peripheral Golgi protein containing a PI-transfer domain, is essential for maintaining the structural and functional integrity of the Golgi apparatus in mammalian cells. Depletion of Nir2 by RNAi leads to substantial inhibition of protein transport from the trans-Golgi network to the plasma membrane, and causes a reduction in the DAG level in the Golgi apparatus. Remarkably, inactivation of cytidine [corrected] 5'-diphosphate (CDP)-choline pathway for phosphatidylcholine biosynthesis restores both effects. These results indicate that Nir2 is involved in maintaining a critical DAG pool in the Golgi apparatus by regulating its consumption via the CDP-choline pathway, demonstrating the interface between secretion from the Golgi and lipid homeostasis.     

Targeting of the GRIP domain to the trans-Golgi network is conserved from protists to animals

The GRIP domain, found in a family of coiled-coil peripheral membrane Golgi proteins, is a specific targeting sequence for the trans-Golgi network of animal cells. In this study we show that a coiled-coil protein with a GRIP domain occurs in the primitive eukaryote, Trypanosoma brucei, and that reporter proteins containing this domain can be used as a marker for the poorly characterized trans Golgi/trans-Golgi network of trypanosomatid parasites. The T. brucei GRIP domain, when fused to the carboxyl terminus of the green fluorescent protein (GFP-TbGRIP), was efficiently localized to the Golgi apparatus of transfected COS cells. Overexpression of GFP-TbGRIP in COS cells displaced the endogenous GRIP protein, GCC1p, from the Golgi apparatus indicating that the trypanosomatid and mammalian GRIP sequences interact with similar membrane determinants. GFP fusion proteins containing either the T. brucei GRIP domain or the human p230 GRIP (p230GRIP) domain were also expressed in the trypanosomatid parasite, Leishmania mexicana, and localized by fluorescence and immuno-electron microscopy to the trans face of the single Golgi apparatus and a short tubule that extended from the Golgi apparatus. Binding of GFP-p230GRIP to Golgi membranes in L. mexicana was abrogated by mutation of a critical tyrosine residue in the p230 GRIP domain. The levels of GFP-GRIP fusion proteins were dramatically reduced in stationary-phase L. mexicana promastigotes, suggesting that specific Golgi trafficking steps may be down-regulated as the promastigotes cease dividing. This study provides a protein marker for the trans-Golgi network of trypanosomatid parasites and suggests that the GRIP domain binds to a membrane component that has been highly conserved in eukaryotic evolution.     

Arabidopsis mannan synthase CSLA9 and glucan synthase CSLC4 have opposite orientations in the Golgi membrane

Several proteins encoded by the cellulose synthase-like (CSL) gene family are known to be processive glycan synthases involved in the synthesis of cell-wall polysaccharides. These include CSLA proteins, which synthesize β-(1→4)-linked mannans found in the walls of many plant species, and CSLC proteins, which are thought to synthesize the β-(1→4)-linked glucan backbone of xyloglucan, an abundant polysaccharide in the primary walls of many plants. CSLA and CSLC proteins are predicted to have multiple membrane spans, and their products (mannan and xyloglucan) accumulate in the Golgi lumen. Knowing where these proteins are located in the cell and how they are orientated in the membrane is important for understanding many aspects of mannan and xyloglucan biosynthesis. In this study, we investigate the subcellular localization and membrane protein topology of CSLA9 and CSLC4, the members of these two families that are most highly expressed in Arabidopsis. CSLA9 and CSLC4 are found predominantly in Golgi membranes, based on co-localization with the known ER/Golgi marker ERD2-YFP. The topology of epitope-tagged proteins was examined using protease protection experiments. Experiments were designed to determine the positions of both the protein termini and the active loop of the CSL proteins investigated. The topology of CSLA9 is characterized by an odd number of transmembrane domains (probably five) and an active site that faces the Golgi lumen. In contrast, CSLC4 has an even number of transmembrane domains (probably six) and an active site that faces the cytosol. The implications of these topologies on various aspects of hemicellulose biosynthesis are discussed.     

Identify Golgi protein types with modified Mahalanobis discriminant algorithm and pseudo amino acid composition

The Golgi apparatus is an important eukaryotic organelle. Successful prediction of Golgi protein types can provide valuable information for elucidating protein functions involved in various biological processes. In this work, a method is proposed by combining a special mode of pseudo amino acid composition (increment of diversity) with the modified Mahalanobis discriminant for predicting Golgi protein types. The benchmark dataset used to train the predictor thus formed contains 95 Golgi proteins in which none of proteins included has ≥40% pairwise sequence identity to any other. The accuracy obtained by the jackknife test was 74.7%, with the ROC curve of 0.772 in identifying cis-Golgi proteins and trans-Golgi proteins. Subsequently, the method was extended to discriminate cis-Golgi network proteins from cis-Golgi network membrane proteins and trans-Golgi network proteins from trans-Golgi network membrane proteins, respectively. The accuracies thus obtained were 76.1% and 83.7%, respectively. These results indicate that our method may become a useful tool in the relevant areas. As a user-friendly web-server, the predictor is freely accessible at http://immunet.cn/SubGolgi/.     

Kinetics of intracellular transport and sorting of lysosomal membrane and plasma membrane proteins

We have used monospecific antisera to two lysosomal membrane glycoproteins, lgp120 and a similar protein, lgp110, to compare the biosynthesis and intracellular transport of lysosomal membrane components, plasma membrane proteins, and lysosomal enzymes. In J774 cells and NRK cells, newly synthesized lysosomal membrane and plasma membrane proteins (the IgG1/IgG2b Fc receptor or influenza virus hemagglutinin) were transported through the Golgi apparatus (defined by acquisition of resistance to endo-beta-N-acetylglucosaminidase H) with the same kinetics (t1/2 = 11-14 min). In addition, immunoelectron microscopy of normal rat kidney cells showed that lgp120 and vesicular stomatitis virus G-protein were present in the same Golgi cisternae demonstrating that lysosomal and plasma membrane proteins were not sorted either before or during transport through the Golgi apparatus. To define the site at which sorting occurred, we compared the kinetics of transport of lysosomal and plasma membrane proteins and a lysosomal enzyme to their respective destinations. Newly synthesized proteins were detected in dense lysosomes (lgp's and beta-glucuronidase) or on the cell surface (Fc receptor or hemagglutinin) after the same lag period (20-25 min), and accumulated at their final destinations with similar kinetics (t1/2 = 30-45 min), suggesting that these two lgp's are not transported to the plasma membrane before reaching lysosomes. This was further supported by measurements of the transport of membrane-bound endocytic markers from the cell surface to lysosomes, which exhibited additional lag periods of 5-15 min and half-times of 1.5-2 h. The time required for transport of newly synthesized plasma membrane proteins to the cell surface, and for the transport of plasma membrane markers from the cell surface to lysosomes would appear too long to account for the rapid transport of lgp's from the Golgi apparatus to lysosomes. Thus, the observed kinetics suggest that lysosomal membrane proteins are sorted from plasma membrane proteins at a post-Golgi intracellular site, possibly the trans Golgi network, before their delivery to lysosomes.     

Genome-Wide Screening of Genes Required for Glycosylphosphatidylinositol Biosynthesis

Glycosylphosphatidylinositol (GPI) is synthesized and transferred to proteins in the endoplasmic reticulum (ER). GPI-anchored proteins are then transported from the ER to the plasma membrane through the Golgi apparatus. To date, at least 17 steps have been identified to be required for the GPI biosynthetic pathway. Here, we aimed to establish a comprehensive screening method to identify genes involved in GPI biosynthesis using mammalian haploid screens. Human haploid cells were mutagenized by the integration of gene trap vectors into the genome. Mutagenized cells were then treated with a bacterial pore-forming toxin, aerolysin, which binds to GPI-anchored proteins for targeting to the cell membrane. Cells that showed low surface expression of CD59, a GPI-anchored protein, were further enriched for. Gene trap insertion sites in the non-selected population and in the enriched population were determined by deep sequencing. This screening enriched 23 gene regions among the 26 known GPI biosynthetic genes, which when mutated are expected to decrease the surface expression of GPI-anchored proteins. Our results indicate that the forward genetic approach using haploid cells is a useful and powerful technique to identify factors involved in phenotypes of interest.     

Regulation of Golgi turnover by CALCOCO1-mediated selective autophagy

The Golgi complex is essential for the processing, sorting, and trafficking of newly synthesized proteins and lipids. Golgi turnover is regulated to meet different cellular physiological demands. The role of autophagy in the turnover of Golgi, however, has not been clarified. Here we show that CALCOCO1 binds the Golgi-resident palmitoyltransferase ZDHHC17 to facilitate Golgi degradation by autophagy during starvation. Depletion of CALCOCO1 in cells causes expansion of the Golgi and accumulation of its structural and membrane proteins. ZDHHC17 itself is degraded by autophagy together with other Golgi membrane proteins such as TMEM165. Taken together, our data suggest a model in which CALCOCO1 mediates selective Golgiphagy to control Golgi size and morphology in eukaryotic cells via its interaction with ZDHHC17.     

Molecular mechanisms and regulation of ceramide transport

De novo biosynthesis of sphingolipids begins in the endoplasmic reticulum (ER) and continues in the Golgi apparatus and plasma membrane. A crucial step in sphingolipid biosynthesis is the transport of ceramide by vesicular and non-vesicular mechanisms from its site of synthesis in the ER to the Golgi apparatus. The recent discovery of the ceramide transport protein CERT has revealed a novel pathway for the delivery of ceramide to the Golgi apparatus for sphingomyelin (SM) synthesis. In addition to a ceramide-binding START domain, CERT has FFAT (referring to two phenylalanines [FF] in an acidic tract) and pleckstrin homology (PH) domains that recognize the ER integral membrane protein VAMP-associated protein (VAP) and Golgi-associated PtdIns 4-phosphate, respectively. Mechanisms for vectorial transport involving dual-organellar targeting and sites of deposition of ceramide in the Golgi apparatus are proposed. Similar Golgi-ER targeting motifs are also present in the oxysterol-binding protein (OSBP), which regulates ceramide transport and SM synthesis in an oxysterol-dependent manner. Consequently, this emerges as a potential mechanism for integration of sphingolipid and cholesterol metabolism. The identification of organellar targeting motifs in other related lipid-binding/transport proteins indicate that concepts learned from the study of ceramide transport can be applied to other lipid transport processes.     

Structural integrity of the Golgi stack is essential for normal secretory functions of rat parotid acinar cells: effects of brefeldin A and okadaic acid.

We examined the effects of specific inhibitors, brefeldin A (BFA) and okadaic acid (OA), on the ultrastructural organization of the Golgi apparatus and distributions of amylase, Golgi-associated proteins, and cathepsin D in the rat parotid acinar cells. BFA induced a rapid regression of the Golgi stack into rudimentary Golgi clusters composed of tubulovesicules, in parallel with a redistribution of the Golgi-resident proteins and a coat protein (beta-COP) into the region of the rough endoplasmic reticulum (rER) or cytosol. The rapid disruption of the Golgi stack could also be induced by the effect of OA. However, redistribution of the Golgi proteins in rER or cytosol could not be observed and beta-COP was not dispersed but was retained on the rudimentary Golgi apparatus. These findings suggested that the mechanism of OA in inducing degeneration of the Golgi stack was markedly different from that of BFA. In addition, missorting of amylase, a Golgi protein, and cathepsin D into incorrect transport pathways is apparent in the course of the disruption of the Golgi stack by OA. These Golgi-disrupting effects are reversible and the reconstruction of the stacked structure of the Golgi apparatus started immediately after the removal of inhibitors. In the recovery processes, missorting was also observed until the integrated structure of the Golgi apparatus was completely reconstructed. This suggested that the integrated structure of the Golgi apparatus was quite necessary for the occurrence of normal secretory events, including proper sorting of molecules.     

Evidence that the entire Golgi apparatus cycles in interphase HeLa cells: sensitivity of Golgi matrix proteins to an ER exit block.

We tested whether the entire Golgi apparatus is a dynamic structure in interphase mammalian cells by assessing the response of 12 different Golgi region proteins to an endoplasmic reticulum (ER) exit block. The proteins chosen spanned the Golgi apparatus and included both Golgi glycosyltransferases and putative matrix proteins. Protein exit from ER was blocked either by microinjection of a GTP-restricted Sar1p mutant protein in the presence of a protein synthesis inhibitor, or by plasmid-encoded expression of the same dominant negative Sar1p. All Golgi region proteins examined lost juxtanuclear Golgi apparatus-like distribution as scored by conventional and confocal fluorescence microscopy in response to an ER exit block, albeit with a differential dependence on Sar1p concentration. Redistribution of GalNAcT2 was more sensitive to low Sar1p(dn) concentrations than giantin or GM130. Redistribution was most rapid for p27, COPI, and p115. Giantin, GM130, and GalNAcT2 relocated with approximately equal kinetics. Distinct ER accumulation could be demonstrated for all integral membrane proteins. ER-accumulated Golgi region proteins were functional. Photobleaching experiments indicated that Golgi-to-ER protein cycling occurred in the absence of any ER exit block. We conclude that the entire Golgi apparatus is a dynamic structure and suggest that most, if not all, Golgi region-integral membrane proteins cycle through ER in interphase cells.     

The dynamics of engineered resident proteins in the mammalian Golgi complex relies on cisternal maturation

After leaving the endoplasmic reticulum, secretory proteins traverse several membranous transport compartments before reaching their destinations. How they move through the Golgi complex, a major secretory station composed of stacks of membranous cisternae, is a central yet unsettled issue in membrane biology. Two classes of mechanisms have been proposed. One is based on cargo-laden carriers hopping across stable cisternae and the other on “maturing” cisternae that carry cargo forward while progressing through the stack. A key difference between the two concerns the behavior of Golgi-resident proteins. Under stable cisternae models, Golgi residents remain in the same cisterna, whereas, according to cisternal maturation, Golgi residents recycle from distal to proximal cisternae via retrograde carriers in synchrony with cisternal progression. Here, we have engineered Golgi-resident constructs that can be polymerized at will to prevent their recycling via Golgi carriers. Maturation models predict the progress of such polymerized residents through the stack along with cargo, but stable cisternae models do not. The results support the cisternal maturation mechanism.