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At least 19 recordsLinked to original sources
PubMed2003

Dispersal of Golgi matrix proteins during mitotic Golgi disassembly.

During mitosis, the mammalian Golgi disassembles into numerous vesicles and larger membrane structures referred to as clusters or remnants. Following mitosis, the vesicles and clusters reassemble to form an intact Golgi in each daughter cell. One model of Golgi biogenesis states that Golgi matrix proteins remain assembled in mitotic clusters and then serve as a template for Golgi reassembly. To test this idea, we performed a 3D-computational analysis of mitotic cells to determine the extent to which these proteins remain in mitotic clusters. As a control we used brefeldin A-induced Golgi disassembly which causes dispersal of Golgi enzymes, but leaves matrix proteins in remnant structures. Unlike brefeldin A-treated cells, in which matrix proteins were clearly sorted from non-matrix proteins, we observed extensive dispersal of matrix proteins in metaphase cells with no evidence of differential sorting of these proteins from other Golgi proteins. The extensive disassembly of matrix proteins argues against their participation in a stable template and supports a self-assembly mode of Golgi biogenesis.

Animals↗
PubMed2000

The mitotic phosphorylation cycle of the cis-Golgi matrix protein GM130.

The cis-Golgi matrix protein GM130 is phosphorylated in mitosis on serine 25. Phosphorylation inhibits binding to p115, a vesicle-tethering protein, and has been implicated as an important step in the mitotic Golgi fragmentation process. We have generated an antibody that specifically recognizes GM130 phosphorylated on serine 25, and used this antibody to study the temporal regulation of phosphorylation in vivo. GM130 is phosphorylated in prophase as the Golgi complex starts to break down, and remains phosphorylated during further breakdown and partitioning of the Golgi fragments in metaphase and anaphase. In telophase, GM130 is dephosphorylated as the Golgi fragments start to reassemble. The timing of phosphorylation and dephosphorylation correlates with the dissociation and reassociation of p115 with Golgi membranes. GM130 phosphorylation and p115 dissociation appear specific to mitosis, since they are not induced by several drugs that trigger nonmitotic Golgi fragmentation. The phosphatase responsible for dephosphorylation of mitotic GM130 was identified as PP2A. The active species was identified as heterotrimeric phosphatase containing the Balpha regulatory subunit, suggesting a role for this isoform in the reassembly of mitotic Golgi membranes at the end of mitosis.

Animals↗
PubMed2004

Dynamics of Golgi matrix proteins after the blockage of ER to Golgi transport.

When the ER to Golgi transport is blocked by a GTP-restricted mutant of Sar1p (H79G) in NRK-52E cells, most Golgi resident proteins are transported back into the ER. In contrast, the cis-Golgi matrix proteins GM130 and GRASP65 are retained in punctate cytoplasmic structures, namely Golgi remnants. Significant amounts of the medial-Golgi matrix proteins golgin-45, GRASP55 and giantin are retained in the Golgi remnants, but a fraction of these proteins relocates to the ER. Golgin-97, a candidate trans-Golgi network matrix protein, is retained in Golgi remnant-like structures, but mostly separated from GM130 and GRASP65. Interestingly, most Sec13p, a COPII component, congregates into larger cytoplasmic clusters soon after the microinjection of Sar1p(H79G), and these move to accumulate around the Golgi apparatus. Sec13p clusters remain associated with Golgi remnants after prolonged incubation. Electron microscopic analysis revealed that Golgi remnants are clusters of larger vesicles with smaller vesicles, many of which are coated. GM130 is mainly associated with larger vesicles and Sec13p with smaller coated vesicles. The Sec13p clusters disperse when p115 binding to the Golgi apparatus is inhibited. These results suggest that cis-Golgi matrix proteins resist retrograde transport flow and stay as true residents in Golgi remnants after the inhibition of ER to Golgi transport.

Autoantigens↗
PubMed2001

Direct targeting of cis-Golgi matrix proteins to the Golgi apparatus.

The targeting route of newly synthesized GM130 and GRASP65 to the Golgi apparatus was investigated by three different approaches. First, localization of pulse labeled GM130 and GRASP65 in normal rat kidney (NRK) cells was traced by subcellular fractionation followed by immunoprecipitation. Immediately after the pulse labeling, GM130 and GRASP65 were found in the Golgi but not in the endoplasmic reticulum (ER) membrane fractions, whereas a control Golgi membrane protein was still found in the ER membrane fractions. Second, epitope tagged GM130 and GRASP65 were expressed in NRK cells by plasmid microinjection into the nuclei and their localization was analyzed by immunofluorescence. When ER to Golgi transport was inhibited by prior microinjection of a GTP-restricted mutant of Sar1 protein into the cytosol, the expressed GM130 and GRASP65 showed clear Golgi localization. Last, binding of GM130 and GRASP65 to the membranes was analyzed in vitro. In vitro synthesized GM130 and GRASP65 specifically bound to purified Golgi membranes but not to microsomal membranes. The bound GM130 and GRASP65 were found to form a complex with pre-existing counterparts on the Golgi membrane. These results strongly suggested that GM130 and GRASP65 are directly targeted to the Golgi membrane without initial assembly on the ER and subsequent vesicular transport to the Golgi apparatus.

Animals↗
PubMed1997

The vesicle docking protein p115 binds GM130, a cis-Golgi matrix protein, in a mitotically regulated manner.

The docking of transport vesicles with their target membrane is thought to be mediated by p115. We show here that GM130, a cis-Golgi matrix protein, interacts specifically with p115 and so could provide a membrane docking site. Deletion analysis showed that the N-terminus binds to p115, whereas the C-terminus binds to Golgi membranes. Mitotic phosphorylation of GM130 or a peptide derived from the N-terminus prevented binding to p115. The peptide also inhibited the NSF- but not the p97-dependent reassembly of Golgi cisternae from mitotic fragments, unless it was mitotically phosphorylated. Together, these data provide a molecular explanation for the COPI-mediated fragmentation of the Golgi apparatus at the onset of mitosis.

Amino Acid Sequence↗
PubMed2005

Cis-Golgi matrix proteins move directly to endoplasmic reticulum exit sites by association with tubules.

The role of cis-medial Golgi matrix proteins in retrograde traffic is poorly understood. We have used imaging techniques to understand the relationship between the cis-medial Golgi matrix and transmembrane proteins during retrograde traffic in control and brefeldin A (BFA)-treated cells. All five of the cis-medial matrix proteins tested were associated with retrograde tubules within 2-3 min of initiation of tubule formation. Then, at later time points (3-10 min), transmembrane proteins are apparent in the same tubules. Strikingly, both the matrix proteins and the transmembrane proteins moved directly to endoplasmic reticulum (ER) exit sites labeled with p58 and Sec13, and there seemed to be a specific interaction between the ER exit sites and the tips or branch points of the tubules enriched for the matrix proteins. After the initial interaction, Golgi matrix proteins accumulated rapidly (5-10 min) at ER exit sites, and Golgi transmembrane proteins accumulated at the same sites approximately 2 h later. Our data suggest that Golgi cis-medial matrix proteins participate in Golgi-to-ER traffic and play a novel role in tubule formation and targeting.

Animals↗
PubMed2003

Characterization of Grp1p, a novel cis-Golgi matrix protein.

A high copy suppressor screen with sec34-2, a temperature-sensitive mutant defective in the late stages of ER to Golgi transport, has resulted in the identification of a novel gene called GRP1 (also called RUD3). GRP1 encodes a hydrophilic yeast protein related to the mammalian Golgi matrix protein golgin-160. A large portion of the protein is predicted to form a coiled-coil structure. Although GRP1 is not essential for growth, the loss of Grp1p results in a growth defect at high temperature. GRP1 genetically interacts with several genes involved in vesicle targeting/fusion stages of ER to Golgi transport. Despite these interactions, pulse chase analysis using Grp1p-depleted cells did not reveal a significant delay in the transit of the vacuolar protease carboxypeptidase Y. Grp1p-depleted cells efficiently secreted invertase which was underglycosylated, suggesting some disturbance of Golgi function. Grp1p-GFP predominantly colocalizes with the cis-Golgi marker Och1p. Despite lacking a signal peptide and a significant stretch of hydrophobic amino acids, Grp1p pellets with membranes. It is extracted with 1M NaCl or 0.1M Na(2)CO(3) (pH 11.0), but is surprisingly insoluble in 1% Triton X-100. Grp1p does not recycle to the ER when forward transport is blocked and a cis-Golgi marker (Och1p-HA), but not a trans-Golgi marker (Chs5p-HA), became dispersed in grp1 Delta cells after 1.5h incubation at 38.5 degrees C. Together, these data suggest that Grp1p is a novel matrix protein that is involved in the structural organization of the cis-Golgi.

Carrier Proteins↗
PubMed2004

YSK1 is activated by the Golgi matrix protein GM130 and plays a role in cell migration through its substrate 14-3-3zeta.

The Golgi apparatus has long been suggested to be important for directing secretion to specific sites on the plasma membrane in response to extracellular signaling events. However, the mechanisms by which signaling events are coordinated with Golgi apparatus function remain poorly understood. Here, we identify a scaffolding function for the Golgi matrix protein GM130 that sheds light on how such signaling events may be regulated. We show that the mammalian Ste20 kinases YSK1 and MST4 target to the Golgi apparatus via the Golgi matrix protein GM130. In addition, GM130 binding activates these kinases by promoting autophosphorylation of a conserved threonine within the T-loop. Interference with YSK1 function perturbs perinuclear Golgi organization, cell migration, and invasion into type I collagen. A biochemical screen identifies 14-3-3zeta as a specific substrate for YSK1 that localizes to the Golgi apparatus, and potentially links YSK1 signaling at the Golgi apparatus with protein transport events, cell adhesion, and polarity complexes important for cell migration.

14-3-3 Proteins↗
PubMed1998

Cdc2 kinase directly phosphorylates the cis-Golgi matrix protein GM130 and is required for Golgi fragmentation in mitosis.

Mitotic fragmentation of the Golgi apparatus can be largely explained by disruption of the interaction between GM130 and the vesicle-docking protein p115. Here we identify a single serine (Ser-25) in GM130 as the key phosphorylated target and Cdc2 as the responsible kinase. MEK1, a component of the MAP kinase signaling pathway recently implicated in mitotic Golgi fragmentation, was not required for GM130 phosphorylation or mitotic fragmentation either in vitro or in vivo. We propose that Cdc2 is directly involved in mitotic Golgi fragmentation and that signaling via MEK1 is not required for this process.

Autoantigens↗
PubMed2006

Brain-type creatine kinase BB-CK interacts with the Golgi Matrix Protein GM130 in early prophase.

Creatine kinase (CK) isoenzymes are essential for storing, buffering and intracellular transport of "energy-rich" phosphate compounds in tissues with fluctuating high energy demand such as muscle, brain and other tissues and cells where CK is expressed. In brain and many non-muscle cells, ubiquitous cytosolic "brain-type" BB-CK and ubiquitous mitochondrial CK (uMtCK) act as components of a phosphocreatine shuttle to maintain cellular energy pools and distribute energy flux. To date, still relatively little is known about direct coupling of functional dimeric BB-CK with other partner proteins or enzymes that are important for cell function. Using a global yeast two-hybrid (Y2H) screen with monomeric B-CK as bait and a representative brain cDNA library to search for interaction partners of B-CK with proteins of the brain, we repeatedly identified the cis-Golgi Matrix protein (GM130) as recurrent interacting partner of B-CK. Since HeLa cells also express both BB-CK and GM130, we subsequently used this cellular model system to verify and characterize the BB-CK-GM130 complex by GST-pulldown experiments, as well as by in vivo co-localization studies with confocal microscopy. Using dividing HeLa cells, we report here for the first time that GM130 and BB-CK co-localize specifically in a transient fashion during early prophase of mitosis, when GM130 plays an important role in Golgi fragmentation that starts also at early prophase. These data may shed new light on BB-CK function for energy provision for Golgi-fragmentation that is initiated by cell signalling cascades in the early phases of mitosis.

Amino Acids↗
PubMed2005

Identification and characterization of AtCASP, a plant transmembrane Golgi matrix protein.

Golgins are a family of coiled-coil proteins that are associated with the Golgi apparatus. They are necessary for tethering events in membrane fusion and may act as structural support for Golgi cisternae. Here we report on the identification of an Arabidopsis golgin which is a homologue of CASP, a known transmembrane mammalian and yeast golgin. Similar to its homologues, the plant CASP contains a long N-terminal coiled-coil region protruding into the cytosol and a C-terminal transmembrane domain with amino acid residues which are highly conserved across species. Through fluorescent protein tagging experiments, we show that plant CASP localizes at the plant Golgi apparatus and that the C-terminus of this protein is sufficient for its localization, as has been shown for its mammalian counterpart. In addition, we demonstrate that the plant CASP is able to localize at the mammalian Golgi apparatus. However, mutagenesis of a conserved tyrosine in the transmembrane domain revealed that it is necessary for ER export and Golgi localization of the Arabidopsis CASP in mammalian cells, but is not required for its correct localization in plant cells. These data suggest that mammalian and plant cells have different mechanisms for concentrating CASP in the Golgi apparatus.

Amino Acid Motifs↗
PubMed2001

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.

Autoantigens↗
PubMed2001

Golgi matrix proteins interact with p24 cargo receptors and aid their efficient retention in the Golgi apparatus.

The Golgi apparatus is a highly complex organelle comprised of a stack of cisternal membranes on the secretory pathway from the ER to the cell surface. This structure is maintained by an exoskeleton or Golgi matrix constructed from a family of coiled-coil proteins, the golgins, and other peripheral membrane components such as GRASP55 and GRASP65. Here we find that TMP21, p24a, and gp25L, members of the p24 cargo receptor family, are present in complexes with GRASP55 and GRASP65 in vivo. GRASPs interact directly with the cytoplasmic domains of specific p24 cargo receptors depending on their oligomeric state, and mutation of the GRASP binding site in the cytoplasmic tail of one of these, p24a, results in it being transported to the cell surface. These results suggest that one function of the Golgi matrix is to aid efficient retention or sequestration of p24 cargo receptors and other membrane proteins in the Golgi apparatus.

Animals↗
PubMed2001

The Golgi matrix protein GM130: a specific interacting partner of the small GTPase rab1b.

To detect specific partners of the small Golgi-localized GTPase rab1b we generated rab1b mutants and used them as bait proteins in yeast two-hybrid screens. We isolated several specifically interacting clones. Two of them encode large protein fragments highly homologous to rat GM130 and to human Golgin95. The full-length human GM130 cDNA was cloned and its interaction with rab1b was characterized in detail by yeast two-hybrid and in vitro binding assays. Here we report for the first time that the rab1b protein interacts specifically with GM130 in a GTP-dependent manner and therefore needs the hypervariable regions of the N- and C-termini. We mapped the rab1b binding site of GM130 and provide evidence that it is different to the previously described p115 and Grasp65 binding sites of the GM130 protein.

Amino Acid Sequence↗
PubMed1995

Characterization of a cis-Golgi matrix protein, GM130.

Antisera raised to a detergent- and salt-resistant matrix fraction from rat liver Golgi stacks were used to screen an expression library from rat liver cDNA. A full-length clone was obtained encoding a protein of 130 kD (termed GM130), the COOH-terminal domain of which was highly homologous to a Golgi human auto-antigen, golgin-95 (Fritzler et al., 1993). Biochemical data showed that GM130 is a peripheral cytoplasmic protein that is tightly bound to Golgi membranes and part of a larger oligomeric complex. Predictions from the protein sequence suggest that GM130 is an extended rod-like protein with coiled-coil domains. Immunofluorescence microscopy showed partial overlap with medial- and trans-Golgi markers but almost complete overlap with the cis-Golgi network (CGN) marker, syntaxin5. Immunoelectron microscopy confirmed this location showing that most of the GM130 was located in the CGN and in one or two cisternae on the cis-side of the Golgi stack. GM130 was not re-distributed to the ER in the presence of brefeldin A but maintained its overlap with syntaxin5 and a partial overlap with the ER-Golgi intermediate compartment marker, p53. Together these results suggest that GM130 is part of a cis-Golgi matrix and has a role in maintaining cis-Golgi structure.

Amino Acid Sequence↗
PubMed2002

Golgi matrix protein gene, Golga3/Mea2, rearranged and re-expressed in pachytene spermatocytes restores spermatogenesis in the mouse.

In a transgenic mouse, Golga3/Mea2 gene (human homolog: GOLGA3/golgin-160) was disrupted by a translocation at the site of the transgene integration. Exons 8-24 of the disrupted gene remained intact and formed a fusion gene (DeltaMea2) with the antisense strand of E. coli-derived transgene by means of a cryptic splice signal in there. The protein product of DeltaMea2, virtually a form truncated to 2/3 of the normal size, localized to Golgi apparatus of pachytene spermatocytes and round spermatids. DeltaMea2 expression was specific to the testis, but varied among separate seminiferous tubules. It also showed variation among homozygous individuals from 0.5 to 4.3% of the wild type (wt) level. At the lowest levels, neither spermatids nor spermatozoa were present in the homozygous testes, but when the expression of DeltaMea2 increased to 4.3% of the wt level, high sperm production was restored and a sporadic (1/22) fertile homozygous male was obtained. The earliest apoptotic degeneration of pachytene spermatocytes evidenced at 17 dpp in homozygous testes in some discrete seminiferous tubules was preceded by DeltaMea2 expression in a variegated fashion at 16 dpp. These results consistently indicated that in homozygous testes, the pachytene spermatocytes which failed to express DeltaMea2 may undergo apoptotic degeneration. Golga3/Mea2, and DeltaMea2 in homozygotes, in a certain excessive amount may be important for survival of pachytene spermatocytes in the mouse.

Animals↗
PubMed2005

dGRASP localization and function in the early exocytic pathway in Drosophila S2 cells.

The de novo model for Golgi stack biogenesis predicts that membrane exiting the ER at transitional ER (tER) sites contains and recruits all the necessary molecules to form a Golgi stack, including the Golgi matrix proteins, p115, GM130, and GRASP65/55. These proteins leave the tER sites faster than Golgi transmembrane resident enzymes, suggesting that they act as a template nucleating the formation of the Golgi apparatus. However, the localization of the Golgi matrix proteins at tER sites is only shown under conditions where exit from the ER is blocked. Here, we show in Drosophila S2 cells, that dGRASP, the single Drosophila homologue of GRASP65/55, localizes both to the Golgi membranes and the tER sites at steady state and that the myristoylation of glycine 2 is essential for the localization to both compartments. Its depletion for 96 h by RNAi gave an effect on the architecture of the Golgi stacks in 30% of the cells, but a double depletion of dGRASP and dGM130 led to the quantitative conversion of Golgi stacks into clusters of vesicles and tubules, often featuring single cisternae. This disruption of Golgi architecture was not accompanied by the disorganization of tER sites or the inhibition of anterograde transport. This shows that, at least in Drosophila, the structural integrity of the Golgi stacks is not required for efficient transport. Overall, dGRASP exhibits a dynamic association to the membrane of the early exocytic pathway and is involved in Golgi stack architecture.

Animals↗