GMx33 associates with the trans-Golgi matrix in a dynamic manner and sorts within tubules exiting the Golgi

The trans-Golgi matrix consists of a group of proteins dynamically associated with the trans-Golgi and thought to be involved in anterograde and retrograde Golgi traffic, as well as interactions with the cytoskeleton and maintenance of the Golgi structure. GMx33 is localized to the cytoplasmic face of the trans-Golgi and is also present in a large cytoplasmic pool. Here we demonstrate that GMx33 is dynamically associated with the trans-Golgi matrix, associating and dissociating with the Golgi in seconds. GMx33 can be locked onto the trans-Golgi matrix by GTPgammaS, indicating that its association is regulated in a GTP-dependent manner like several other Golgi matrix proteins. Using live-cell imaging we show that GMx33 exits the Golgi associated with tubules and within these tubules GMx33 segregates from transmembrane proteins followed by fragmentation of the tubules into smaller tubules and vesicles. Within vesicles produced by an in vitro budding reaction, GMx33 remains segregated in a matrixlike tail region that sometimes contains Golgin-245. This trans-matrix often links a few vesicles together. Together these data suggest that GMx33 is a member of the trans-Golgi matrix and offer clues regarding the role of the trans-Golgi matrix in sorting and exit from the Golgi.     

Identification and characterization of a novel alternative splice variant of mouse GMx33alpha/GPP34

We have cloned and characterized a novel splice variant of mouse GMx33alpha/Golgi-associated protein of 34 kDa (GPP34), hereby designated GMx33alphaV/GPP34V. This splice variant skips the second and third exons, and the resulting frame shift generates a stop codon in the fourth exon. GMx33alphaV/GPP34V is comprised of 81 amino acid residues derived from the N-terminal end of the full length protein and corresponds to approximately one-third of the full length GMx33alpha/GPP34 sequence with a calculated molecular mass of 8900. In contrast to GMx33alpha/GPP34 mRNA which is expressed at similar levels in various tissues, GMx33alphaV/GPP34V mRNA was differentially expressed when examined by RT-PCR. Compared to other tissues, skeletal muscle showed relatively strong expression of GMx33alphaV/GPP34V mRNA. This splice variant cDNA was also detected in a human cell line.     

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.     

Study of GOLPH3: a Potential Stress-Inducible Protein from Golgi Apparatus

Although the Golgi apparatus has been studied extensively for over 100 years, the complex structure-function relationships have yet to be elucidated. It is well known that the Golgi complex plays an important role in the transport, processing, sorting, and targeting of numerous proteins and lipids destined for secretion, plasma membrane, and lysosomes. Increasing evidence suggests that the Golgi apparatus is a sensor and common downstream effector of stress signals in cell death pathways. It undergoes disassembly and fragmentation in several neurological disorders. Recent studies indicate that Golgi phosphoprotein 3 (GOLPH3 also known as GPP34/GMx33/MIDAS), a peripheral membrane protein of trans-Golgi network, represents an exciting new class of oncoproteins involved in cell signal transduction and is potentially mobilized by stress. In this review, we focus on the importance of GOLPH3 in vesicular trafficking, Golgi architecture maintenance, receptor sorting, protein glycosylation, and further discuss its potential in signal sensing in stress response.     

CHARACTERIZATION OF MULTIPLE FORMS OF BRAIN TUBULIN SUBUNITS

Abstract— Microtubular protein was isolated from rat forebrain by biochemical purification (ammonium sulfate precipitation followed by DEAE cellulose chromatography) or by two cycles of aggregation-disaggregation. The protein subunit structure was examined on two-dimensional electrophoretograms: first dimension, urea isoelectric focusing gel; second dimension, sodium dodecyl sulfate exponential acrylamide slab gel. Two forms of α tubulin were separated in the second dimension on the basis of different rates of migration (α and α₂). Each of these species was further separated into at least three forms with different isoelectric points. β Tubulin was separated into a minor species (BI) and a major species β₂). Multiple subunits were observed using protein from either purification method and in a two-dimensional electrophoretogram of total supernatant proteins from rat brain. Separation and visualization of multiple forms of α and β tubulin is consistent with reports that provide evidence for post-translational modification of these proteins.     

The Sec1/Munc18 protein, Vps33p, functions at the endosome and the vacuole of Saccharomyces cerevisiae

The Sec1/Munc18 (SM) family of proteins is thought to impart compartmental specificity to vesicle fusion reactions. Here we report characterization of Vps33p, an SM family member previously thought to act exclusively at the vacuolar membrane with the vacuolar syntaxin Vam3p. Vacuolar morphology of vps33Delta cells resembles that of cells lacking both Vam3p and the endosomal syntaxin Pep12p, suggesting that Vps33p may function with these syntaxins at the vacuole and the endosome. Consistent with this, vps33 mutants secrete the Golgi precursor form of the vacuolar hydrolase CPY into the medium. We also demonstrate that Vps33p acts at other steps, for vps33 mutants show severe defects in endocytosis at the late endosome. At the endosome, Vps33p and other class C members exist as a complex with Vps8p, a protein previously known to act in transport between the late Golgi and the endosome. Vps33p also interacts with Pep12p, a known interactor of the SM protein Vps45p. High copy PEP7/VAC1 suppresses vacuolar morphology defects of vps33 mutants. These findings demonstrate that Vps33p functions at multiple trafficking steps and is not limited to action at the vacuolar membrane. This is the first report demonstrating the involvement of a single syntaxin with two SM proteins at the same organelle.     

Monensin and brefeldin A differentially affect the phosphorylation and sulfation of secretory proteins.

Chromogranin B and secretogranin II, two members of the granin family, are known to be post-translationally modified by the addition of O-linked carbohydrates to serine and/or threonine, phosphate to serine and threonine, and sulfate to carbohydrate and tyrosine residues. In the present study, chromogranin B and secretogranin II were used as model proteins to investigate in which subcompartment of the Golgi complex secretory proteins become phosphorylated. Monensin, a drug known to block the transport from the medial to the trans cisternae of the Golgi stack, inhibited the phosphorylation of the granins, indicating that this modification occurred distal to the medial Golgi. Monensin also blocked the addition of galactose to O-linked carbohydrates and the sulfation of the granins, confirming previous data that these modifications take place in the trans Golgi. To distinguish, within the trans Golgi, between the trans cisternae of the Golgi stack and the trans Golgi network, we made use of the previous observation that brefeldin A results in the redistribution to the endoplasmic reticulum of membrane-bound enzymes of the trans cisternae of the Golgi stack, but not of the trans Golgi network. Brefeldin A treatment abolished granin sulfation but resulted in the accumulation of phosphorylated and galactosylated granins. Differential effects of brefeldin A on membranes of the Golgi stack versus the trans Golgi network were also observed by immunofluorescence analysis of marker proteins specific for either compartment. Our results suggest that the phosphorylation of secretory proteins, like their galactosylation, largely occurs in the trans cisternae of the Golgi stack, whereas the sulfation of secretory proteins on both carbohydrate and tyrosine residues takes place selectively in the trans Golgi network.     

Golgi localization of glycosyltransferases requires a Vps74p oligomer

The mechanism of glycosyltransferase localization to the Golgi apparatus is a long-standing question in secretory cell biology. All Golgi glycosyltransferases are type II membrane proteins with small cytosolic domains that contribute to Golgi localization. To date, no protein has been identified that recognizes the cytosolic domains of Golgi enzymes and contributes to their localization. Here, we report that yeast Vps74p directly binds to the cytosolic domains of cis and medial Golgi mannosyltransferases and that loss of this interaction correlates with loss of Golgi localization of these enzymes. We have solved the X-ray crystal structure of Vps74p and find that it forms a tetramer, which we also observe in solution. Deletion of a critical structural motif disrupts tetramer formation and results in loss of Vps74p localization and function. Vps74p is highly homologous to the human GMx33 Golgi matrix proteins, suggesting a conserved function for these proteins in the Golgi enzyme localization machinery.     

Establishing a Role for the GTPase Ypt1p at the Late Golgi

GTPases of the Rab family cycle between an inactive (GDP‐bound) and active (GTP‐bound) conformation. The active form of the Rab regulates a variety of cellular functions via multiple effectors. Guanine nucleotide exchange factors (GEFs) activate Rabs by accelerating the exchange of GDP for GTP, while GTPase activating proteins (GAPs) inactivate Rabs by stimulating the hydrolysis of GTP. The GTPase Ypt1p is required for endoplasmic reticulum (ER)–Golgi and intra‐Golgi traffic in the yeast Saccharomyces cerevisiae. Recent findings, however, have shown that Ypt1p GEF, GAP and an effector are all required for traffic from the early endosome to the Golgi. Here we describe a screen for ypt1 mutants that block traffic from the early endosome to the late Golgi, but not general secretion. This screen has led to the identification of a collection of recessive and dominant mutants that block traffic from the early endosome. While it has long been known that Ypt1p regulates the flow of biosynthetic traffic into the cis side of the Golgi, these findings have established a role for Ypt1p in the regulation of early endosome–Golgi traffic. We propose that Ypt1p regulates the flow of traffic into the cis and trans side of the Golgi via multiple effectors.     

Rab9 functions in transport between late endosomes and the trans Golgi network.

Rab proteins represent a large family of ras-like GTPases that regulate distinct vesicular transport events at the level of membrane targeting and/or fusion. We report here the primary sequence, subcellular localization and functional activity of a new member of the rab protein family, rab9. The majority of rab9 appears to be located on the surface of late endosomes. Rab9, purified from Escherichia coli strains expressing this protein, could be prenylated in vitro in the presence of cytosolic proteins and geranylgeranyl diphosphate. In vitro-prenylated rab9 protein, but not C-terminally truncated rab9, stimulated the transport of mannose 6-phosphate receptors from late endosomes to the trans Golgi network in a cell-free system that reconstitutes this transport step. Rab7, a related rab protein that is also localized to late endosomes, was inactive in the in vitro transport assay, despite its efficient prenylation and capacity to bind and hydrolyze GTP. These results strongly suggest that rab9 functions in the transport of mannose 6-phosphate receptors between late endosomes and the trans Golgi network. Moreover, our results confirm the observation that a given organelle may bear multiple rab proteins with different biological functions.     

O-sulfonation of serine and threonine: mass spectrometric detection and characterization of a new posttranslational modification in diverse proteins throughout the eukaryotes

Protein sulfonation on serine and threonine residues is described for the first time. This post-translational modification is shown to occur in proteins isolated from organisms representing a broad span of eukaryote evolution, including the invertebrate mollusk Lymnaea stagnalis, the unicellular malaria parasite Plasmodium falciparum, and humans. Detection and structural characterization of this novel post-translational modification was carried out using liquid chromatography coupled to electrospray tandem mass spectrometry on proteins including a neuronal intermediate filament and a myosin light chain from the snail, a cathepsin-C-like enzyme from the parasite, and the cytoplasmic domain of the human orphan receptor tyrosine kinase Ror-2. These findings suggest that sulfonation of serine and threonine may be involved in multiple functions including protein assembly and signal transduction.     

The plant mitochondrial proteome

The plant mitochondrial proteome might contain as many as 2000-3000 different gene products, each of which might undergo post-translational modification. Recent studies using analytical methods, such as one-, two- and three-dimensional gel electrophoresis and one- and two-dimensional liquid chromatography linked on-line with tandem mass spectrometry, have identified >400 mitochondrial proteins, including subunits of mitochondrial respiratory complexes, supercomplexes, phosphorylated proteins and oxidized proteins. The results also highlight a range of new mitochondrial proteins, new mitochondrial functions and possible new mechanisms for regulating mitochondrial metabolism. More than 70 identified proteins in Arabidopsis mitochondrial samples lack similarity to any protein of known function. In some cases, unknown proteins were found to form part of protein complexes, which allows a functional context to be defined for them. There are indications that some of these proteins add novel activities to mitochondrial protein complexes in plants.     

Targeting of proteins to the Golgi apparatus

 The proteins that reside in the Golgi carry out functions associated with post-translational modifications, including glycosylation and proteolytic processing, membrane transport, recycling of endoplasmic reticulum proteins and maintenance of the structural organisation of the organelle itself. The latter includes Golgi stacking, interconnections between stacks and the microtubule-dependent positioning of the organelle within the cell. There are a number of distinct groups of Golgi membrane proteins, including glycosyltransferases, recycling trans-Golgi network (TGN) proteins, peripheral membrane proteins and receptors. Considerable effort has been directed at understanding the basis of the localisation of Golgi glycosyltransferases and recycling TGN proteins; in both cases there is increasing evidence that multiple signals may be involved in their specific localisation. A number of models for the Golgi retention of glycosyltransferases have been proposed including oligomerisation, lipid-mediated sorting and intra-Golgi retrograde transport. More information is required to determine the contribution of each of these potential mechanisms in the targeting of different glycosyltransferases. Future work is also likely to focus on the relationship between the localisation of resident Golgi proteins and the maintenance of Golgi structure. Accepted: 15 October 1997     

Proteomics characterization of abundant Golgi membrane proteins

A mass spectrometric analysis of proteins partitioning into Triton X-114 from purified hepatic Golgi apparatus (84% purity by morphometry, 122-fold enrichment over the homogenate for the Golgi marker galactosyl transferase) led to the unambiguous identification of 81 proteins including a novel Golgi-associated protein of 34 kDa (GPP34). The membrane protein complement was resolved by SDS-polyacrylamide gel electrophoresis and subjected to a hierarchical approach using delayed extraction matrix-assisted laser desorption ionization mass spectrometry characterization by peptide mass fingerprinting, tandem mass spectrometry to generate sequence tags, and Edman sequencing of proteins. Major membrane proteins corresponded to known Golgi residents, a Golgi lectin, anterograde cargo, and an abundance of trafficking proteins including KDEL receptors, p24 family members, SNAREs, Rabs, a single ARF-guanine nucleotide exchange factor, and two SCAMPs. Analytical fractionation and gold immunolabeling of proteins in the purified Golgi fraction were used to assess the intra-Golgi and total cellular distribution of GPP34, two SNAREs, SCAMPs, and the trafficking proteins GBF1, BAP31, and alpha(2)P24 identified by the proteomics approach as well as the endoplasmic reticulum contaminant calnexin. Although GPP34 has never previously been identified as a protein, the localization of GPP34 to the Golgi complex, the conservation of GPP34 from yeast to humans, and the cytosolically exposed location of GPP34 predict a role for a novel coat protein in Golgi trafficking.     

Syntaxin 16: Unraveling cellular physiology through a ubiquitous SNARE molecule

Syntaxin 16 (Syx16) is member of the soluble N‐ethylmaleimide sensitive factor attachment protein receptor (SNARE) family of molecules that functions in membrane fusion in eukaryotic cells. A rather ubiquitously expressed, tail‐anchored membrane protein localized mainly at the trans‐Golgi network (TGN), it mediates primarily retrograde endosomal‐TGN transport. In spite of its ubiquitous expression, Syx16 has specific and interesting roles in the physiology of specialized cells, including Glut4 dynamics, dendritic outgrowth‐related membrane traffic, and cytokinesis. We discussed these physiological functions of Syx16 in the light of what is known of its subcellular localization, vesicular trafficking pathways involved, cognate SNARE partners and other interacting proteins. Further, we speculate on some possible pathophysiological roles of Syx16. J. Cell. Physiol. 225: 326–332, 2010. © 2010 Wiley‐Liss, Inc.     

Differential proteome analysis of mature and germinated seeds of Magnolia sieboldii K. Koch

Key message

This is the first reported proteomic analysis to study the dormancy breaking of Magnolia sieboldii seeds. Our results provide a fundamental reference for further studies on the regulation of protein expression during seed germination.

Abstract

Magnolia sieboldii K. Koch is an ornamental tree. The deep dormancy of its seeds hinders its cultivation for economic purposes. The biochemical basis of the regulation of seed germination remains poorly understood. The present study aimed to identify differentially expressed proteins in germinated seeds of M. sieboldii using polyethylene glycol fractionation. In total, 59 differentially expressed protein spots from two-dimensional gel maps were detected, 33 of which were identified by mass spectrometry. They were assigned to eight functional classes on the basis of their putative biological functions: photosynthesis (3 %), chaperonin/heat shock protein (9 %), protein and amino acid synthesis (9 %), stress/defense (18 %), cytoskeleton structure (3 %), metabolism (18 %), hormone and polyamine (9 %) and storage proteins (31 %). Among the other functions, the effects of plant hormones on seed germination may be one of the most important functions in plant growth. Gibberellins and ethylene positively regulate seed germination. The activities of several hormone-associated proteins possibly influencing seed germination were increased. The characterization of these proteins will be of great help in identifying the molecular mechanism underlying seed germination.     

Cisterna-specific localization of glycosylation-related proteins to the Golgi apparatus

The Golgi apparatus is an intracellular organelle playing central roles in post-translational modification and in the secretion of membrane and secretory proteins. These proteins are synthesized in the endoplasmic reticulum (ER) and transported to the cis-, medial-and trans-cisternae of the Golgi. While trafficking through the Golgi, proteins are sequentially modified with glycan moieties by different glycosyltransferases. Therefore, it is important to analyze the glycosylation function of the Golgi at the level of cisternae. Markers widely used for cis-, medial- and trans-cisternae/trans Golgi network (TGN) in Drosophila are GM130, 120 kDa and Syntaxin16 (Syx16); however the anti-120 kDa antibody is no longer available. In the present study, Drosophila Golgi complex-localized glycoprotein-1 (dGLG1) was identified as an antigen recognized by the anti-120 kDa antibody. A monoclonal anti-dGLG1 antibody suitable for immunohistochemistry was raised in rat. Using these markers, the localization of glycosyltransferases and nucleotide-sugar transporters (NSTs) was studied at the cisternal level. Results showed that glycosyltransferases and NSTs involved in the same sugar modification are localized to the same cisternae. Furthermore, valuable functional information was obtained on the localization of novel NSTs with as yet incompletely characterized biochemical properties.     

Organellar proteomics reveals Golgi arginine dimethylation

The Golgi complex functions to posttranslationally modify newly synthesized proteins and lipids and to sort them to their sites of function. In this study, a stacked Golgi fraction was isolated by classical cell fractionation, and the protein complement (the Golgi proteome) was characterized using multidimensional protein identification technology. Many of the proteins identified are known residents of the Golgi, and 64% of these are predicted transmembrane proteins. Proteins localized to other organelles also were identified, strengthening reports of functional interfacing between the Golgi and the endoplasmic reticulum and cytoskeleton. Importantly, 41 proteins of unknown function were identified. Two were selected for further analysis, and Golgi localization was confirmed. One of these, a putative methyltransferase, was shown to be arginine dimethylated, and upon further proteomic analysis, arginine dimethylation was identified on 18 total proteins in the Golgi proteome. This survey illustrates the utility of proteomics in the discovery of novel organellar functions and resulted in 1) a protein profile of an enriched Golgi fraction; 2) identification of 41 previously uncharacterized proteins, two with confirmed Golgi localization; 3) the identification of arginine dimethylated residues in Golgi proteins; and 4) a confirmation of methyltransferase activity within the Golgi fraction.     

The GRIP domain is a specific targeting sequence for a population of trans-Golgi network derived tubulo-vesicular carriers

Vesicular carriers for intracellular transport associate with unique sets of accessory molecules that dictate budding and docking on specific membrane domains. Although many of these accessory molecules are peripheral membrane proteins, in most cases the targeting sequences responsible for their membrane recruitment have yet to be identified. We have previously defined a novel Golgi targeting domain (GRIP) shared by a family of coiled-coil peripheral membrane Golgi proteins implicated in membrane trafficking. We show here that the docking site for the GRIP motif of p230 is a specific domain of Golgi membranes. By immuno-electron microscopy of HeLa cells stably expressing a green fluorescent protein (GFP)-p230_GRIP fusion protein, we show binding specifically to a subset of membranes of the trans -Golgi network (TGN). Real-time imaging of live HeLa cells revealed that the GFP-p230_GRIP was associated with highly dynamic tubular extensions of the TGN, which have the appearance and behaviour of transport carriers. To further define the nature of the GRIP membrane binding site, in vitro budding assays were performed using purified rat liver Golgi membranes and cytosol from GFP-p230_GRIP-transfected cells. Analysis of Golgi-derived vesicles by sucrose gradient fractionation demonstrated that GFP-p230_GRIP binds to a specific population of vesicles distinct from those labelled for β-COP or γ-adaptin. The GFP-p230_GRIP fusion protein is recruited to the same vesicle population as full-length p230, demonstrating that the GRIP domain is solely proficient as a targeting signal for membrane binding of the native molecule. Therefore, p230 GRIP is a targeting signal for recruitment to a highly selective membrane attachment site on a specific population of trans -Golgi network tubulo-vesicular carriers.