Coated vesicles contain a phosphatidylinositol kinase

When coated vesicles (CVs) are incubated with [gamma-32P]ATP, radioactivity is rapidly incorporated into a compound identified by thin layer chromatography as phosphatidylinositol 4-phosphate. This activity has been identified in CVs isolated from bovine brain as well as from rat liver and chick embryo skeletal muscle. Phosphatidylinositol (PI) kinase is not separated from CVs during agarose electrophoresis, which produces CVs of greater than 95% purity, indicating that the activity present does not derive from contamination. The specific activity of these highly purified CVs was demonstrated to be approximately twice that of synaptic plasma membranes, further ruling out contamination from this source. The PI kinase remains associated with the vesicle upon removal of clathrin and its associated proteins and is solubilized by nonionic detergents, suggesting it is an integral membrane protein. We have been unable to demonstrate the formation of significant amounts of phosphatidylinositol 4,5-bisphosphate in any of our CV preparations. In the presence of exogenous PI, activity is stimulated, with maximal phosphorylation occurring at 0.1 mM. The enzyme appears to be maximally stimulated by 200 mM MgCl2 and 1 mM ATP and is most active at pH 7.25. Calculations indicate that, under optimal conditions, approximately 25 molecules of PIP are produced per CV within 60 s, suggesting that these structures may play an important role in cellular PI metabolism.     

The Golgi-associated hook3 protein is a member of a novel family of microtubule-binding proteins

Microtubules are central to the spatial organization of diverse membrane-trafficking systems. Here, we report that Hook proteins constitute a novel family of cytosolic coiled coil proteins that bind to organelles and to microtubules. The conserved NH(2)-terminal domains of Hook proteins mediate attachment to microtubules, whereas the more divergent COOH-terminal domains mediate the binding to organelles. Human Hook3 bound to Golgi membranes in vitro and was enriched in the cis-Golgi in vivo. Unlike other cis-Golgi-associated proteins, however, a large fraction of Hook3 maintained its juxtanuclear localization after Brefeldin A treatment, indicating a Golgi-independent mechanism for Hook3 localization. Because overexpression of Hook3 caused fragmentation of the Golgi complex, we propose that Hook3 participates in defining the architecture and localization of the mammalian Golgi complex.     

Vear, a novel Golgi-associated protein with VHS and gamma-adaptin "ear" domains

The molecular basis of the selectivity and the details of the vesicle formation in endocytic and secretory pathways are still poorly known and most probably involve as yet unidentified components. Here we describe the cloning, expression, and tissue and cell distribution of a novel protein of 67 kDa (called Vear) that bears homology to several endocytosis-associated proteins in that it has a VHS domain in its N terminus. It is also similar to gamma-adaptin, the heavy subunit of AP-1, in having in its C terminus a typical "ear" domain. In immunofluorescence microscopy, Vear was seen in the Golgi complex as judged by a typical distribution pattern, a distinct colocalization with the Golgi marker gamma-adaptin, and a sensitivity to treatment of cells with brefeldin A. In cell fractionation, Vear partitioned with the post-nuclear membrane fraction. In transfection experiments, hemagglutinin-tagged full-length Vear and truncated Vear lacking the VHS domain assembled on and caused compaction of the Golgi complex. Golgi association without compaction was seen with the ear domain of Vear, whereas the VHS domain alone showed a diffuse membrane- and vesicle-associated distribution. The Golgi association and the bipartite structure along with the differential targeting of its domains suggest that Vear is involved in heterotypic vesicle/suborganelle interactions associated with the Golgi complex. Tissue-specific function of Vear is suggested by its high level of expression in kidney, muscle, and heart.     

Articular cartilage vesicles contain RNA

Small membrane-bound extracellular organelles known as articular cartilage matrix vesicles (ACVs) participate in pathologic mineralization in osteoarthritic articular cartilage. ACVs are also present in normal cartilage, although they have no known functions other than mineralization. Recently, RNA was identified in extracellular vesicles derived from mast cells, suggesting that such vesicles might carry coding information from cell to cell. We found that ACVs from normal porcine and human articular cartilage and primary chondrocyte conditioned media contained 1 μg RNA/80 μg ACV protein. No DNA could be detected. RT-PCR of ACV RNA demonstrated the presence of full length mRNAs for factor XIIIA, type II transglutaminase, collagen II, aggrecan, ANKH and GAPDH. RNA in intact ACVs was resistant to RNase, despite the fact that ACV preparations contained measurable levels of active RNases. Significantly, radiolabeled RNA in ACVs could be transferred to unlabeled chondrocytes by co-incubation and produced changes in levels of chondrocyte enzymes and proteins. The demonstration that ACVs contain mRNAs suggests that they may function to shuttle genetic information between articular cells and indicate novel functions for these structures in articular cartilage.     

Extracellular vesicles produced by Cryptococcus neoformans contain protein components associated with virulence

Cryptococcus neoformans produces vesicles containing its major virulence factor, the capsular polysaccharide glucuronoxylomannan (GXM). These vesicles cross the cell wall to reach the extracellular space, where the polysaccharide is supposedly used for capsule growth or delivered into host tissues. In the present study, we characterized vesicle morphology and protein composition by a combination of techniques including electron microscopy, proteomics, enzymatic activity, and serological reactivity. Secretory vesicles in C. neoformans appear to be correlated with exosome-like compartments derived from multivesicular bodies. Extracellular vesicles manifested various sizes and morphologies, including electron-lucid membrane bodies and electron-dense vesicles. Seventy-six proteins were identified by proteomic analysis, including several related to virulence and protection against oxidative stress. Biochemical tests indicated laccase and urease activities in vesicles. In addition, different vesicle proteins were recognized by sera from patients with cryptococcosis. These results reveal an efficient and general mechanism of secretion of pathogenesis-related molecules in C. neoformans, suggesting that extracellular vesicles function as “virulence bags” that deliver a concentrated payload of fungal products to host effector cells and tissues.     

Amphiphysin, a novel protein associated with synaptic vesicles.

To obtain access to novel proteins of the neuronal synapse, we have raised antisera against proteins of synaptic plasma membranes and used them for immunoscreening brain cDNA expression libraries. One of the newly isolated cDNAs encodes an acidic protein of 75 kDa with a distinct architecture of structural domains and multiple potential phosphorylation sites. Light and electron microscopy employing monospecific antisera raised against the expression product indicate a synapse-specific, presynaptic localization of this protein in many synapses of the chicken and rat nervous system. Its overall distribution in brain is very similar to that of synaptophysin, a ubiquitous protein of synaptic vesicles. In addition to brain, the protein or its mRNA is expressed in adrenal gland and anterior and posterior pituitary, but was not detected in a variety of other tissues. In controlled pore glass chromatography the native protein copurifies with synaptic vesicles and largely remains associated with them under various washing conditions. However, its amino acid sequence is very hydrophilic and it segregates into the aqueous phase in detergent phase partition. An earlier step of synaptic vesicle purification, sucrose cushion centrifugation, separates a vesicle-bound fraction of this protein from an unbound fraction. This seems to be a new, perhaps peripheral, protein of synaptic vesicles for which we propose the name, amphiphysin.     

The plant nuclear envelope protein MAF1 has an additional location at the Golgi and binds to a novel Golgi-associated coiled-coil protein

Tomato MAF1 (LeMAF1) is a plant-specific, nuclear envelope (NE)-associated protein. It is the founding member of a group of WPP domain-containing, NE-associated proteins. This group includes the Arabidopsis WPP family, which is involved in cell division, as well as plant RanGAPs. In addition to its NE localization, LeMAF1 accumulates in speckles in the cytoplasm. Here, we show that the LeMAF1-containing speckles are components of the Golgi apparatus. A novel tomato coiled-coil protein was identified that specifically binds to LeMAF1. Tomato WPP domain-associated protein (LeWAP) interacts in yeast and in vitro through its coiled-coil domain with several WPP-domain containing proteins, including AtRanGAP1 and the WPP family (LeMAF, WPP1 and WPP2). Like LeMAF1, LeWAP is localized at the Golgi. Moreover, we present data showing that Arabidopsis WAP is necessary for the existence of a multi-protein complex containing WPP2. Electronic Supplementary Material Supplementary material is available for this article at .     

Plant cells contain a novel member of the retinoblastoma family of growth regulatory proteins.

The product of the retinoblastoma susceptibility gene (Rb) controls the passage of mammalian cells through G1 phase. Animal virus oncoproteins interact with the Rb protein via an LXCXE motif and disrupt Rb-E2F complexes, driving cells into S-phase. Recently, we found that the RepA protein of a plant geminivirus contains an LXCXE motif that is essential for its function, a finding that predicts the existence of Rb-related proteins in plant cells. Here we report the isolation of a maize cDNA clone encoding a protein (ZmRb1) which, based on structural and functional studies, is closely related to the mammalian Rb family of growth regulatory proteins. ZmRb1 shows a high degree of amino acid conservation when compared with animal Rb members, particularly in the A/B 'pocket' domain, but ZmRb1 has a shorter N-terminal domain. ZmRb1 forms stable complexes with plant LXCXE-containing proteins, e.g. geminivirus RepA protein. Geminivirus DNA replication is reduced in plant cells transfected with plasmids encoding either ZmRb1 or human p130, a member of the Rb family. This suggests that ZmRb1 controls the G1/S transit in plant cells and is consistent with the fact that geminiviruses need an S-phase environment for DNA replication, as animal DNA tumor viruses do. Our results allow the extension of the Rb family of tumor suppressor proteins to plants and have implications on animal and plant strategies for cell growth control.     

Purification and characterization of an alpha-actinin-like protein from porcine kidney

An alpha-actinin-like protein was partially purified from the Triton-insoluble cytoskeleton of porcine kidney by 0.6 M MgCl2 treatment, ammonium sulfate fractionation, DEAE-cellulose chromatography and hydroxyapatite chromatography. Apparent purity of the kidney protein was approximately 90% by quantitative densitometry of Coomassie-stained polyacrylamide gels. The kidney alpha-actinin-like protein is very similar to muscle alpha-actinins by the following criteria: (1) both kidney protein and muscle alpha-actinins bind to F-actin at a similar ratio; (2) both proteins demonstrate no difference in the actomyosin turbidity assay and the ATPase assay for alpha-actinin activity; (3) both native proteins contain a large core of identical molecular weight resistant to trypsin; (4) on two-dimensional gels, both kidney protein and muscle alpha-actinins have similar isoelectric points of 5.9-6.1. However, kidney alpha-actinin-like protein is not identical in every respect with muscle alpha-actinins. Electrophoretic mobility of the kidney protein is slightly greater than that of chicken gizzard alpha-actinin and is identical to that of a component of chicken skeletal muscle alpha-actinin. One-dimensional peptide mappings of the kidney protein and muscle alpha-actinins were significantly different from each other. The interaction between kidney alpha-actinin-like protein and F-actin is sensitive to Ca2+. Similar Ca2+-sensitivity was observed with other non-muscle cell alpha-actinins.     

Plant cells contain calsequestrin

Calsequestrin is a high capacity low affinity Ca2+-binding protein thought to be essential for the function of the intracellular rapid releasable Ca2+ pool of a variety of animal cells. Here we show that two types of plant tissues, cultured Streptanthus tortuosus cells and spinach leaves, contain a form of calsequestrin. In subcellular fractions of S. tortuosus cells, Stains-all staining reveals a metachromatically blue-staining 56,000-Da protein enriched in the microsomal fraction. This protein shares several biochemical characteristics with animal calsequestrin: 1) it changes its apparent molecular weight with the pH; 2) it is able to bind 45Ca2+ on nitrocellulose transfers; and 3) it is recognized by antibodies against canine cardiac calsequestrin. Calsequestrin was also identified in spinach leaves using a direct extraction procedure that was developed for muscle calsequestrin. Thus, our results demonstrate that plant cells contain calsequestrin within a subcellular membrane fraction. These results also suggest that calsequestrin is an ubiquitous protein rather than being limited only to animal cells.     

Identification of a Golgi-associated protein that undergoes mitosis dependent phosphorylation and relocation

By means of a monoclonal antibody (BH3), we have identified a 57-kD protein (p57) that in interphase is restricted largely to the perinuclear region of the cell. Double label immunofluorescence microscopy suggests localization of p57 to the Golgi complex and associated membranous structures. Protease protection experiments and chemical extractability indicate that p57 is a peripheral membrane protein exposed to the cytoplasm. p57 displays unique behavior during mitosis. At the end of G2 or in early prophase, p57 leaves the perinuclear region and accumulates very rapidly within the nucleus, at a time when the nuclear envelope is still intact and before nuclear lamina disassembly. This relocation of p57 coincides with its hyperphosphorylation on serine and threonine residues. After nuclear envelope breakdown p57 becomes uniformly distributed throughout the mitotic cytoplasm until in late telophase when it returns to its perinuclear location and is once again excluded from the nucleus. The behavior of p57 during mitosis suggests that it may play a role in the cellular reorganization evident during mitotic prophase.     

The Golgi-associated protein GRASP is required for unconventional protein secretion during development

During Dictyostelium development, prespore cells secrete acyl-CoA binding protein (AcbA). Upon release, AcbA is processed to generate a peptide called spore differentiation factor-2 (SDF-2), which triggers terminal differentiation of spore cells. We have found that cells lacking Golgi reassembly stacking protein (GRASP), a protein attached peripherally to the cytoplasmic surface of Golgi membranes, fail to secrete AcbA and, thus, produce inviable spores. Surprisingly, AcbA lacks a signal sequence and is not secreted via the conventional secretory pathway (endoplasmic reticulum-Golgi-cell surface). GRASP is not required for conventional protein secretion, growth, and the viability of vegetative cells. Our findings reveal a physiological role of GRASP and provide a means to understand unconventional secretion and its role in development.     

Plant coated vesicles

Abstract. Coated vesicles are organelles frequently encountered in many plant cell types often in association with the plasma membrane, Golgi apparatus, partially coated reticulum and multivesicular bodies. They are readily identified by a characteristic cage or basket composed of interlocking triskelions of the protein clathrin which are bound to the surface of the vesicle membrane. Although their transport function has been well studied and characterized in mammalian systems, the possible importance of coated vesicles as transport organelles in plant cells is only just beginning to be explored. In this review, the authors describe the structure of higher plant coated vesicles and discuss their possible involvement in the endocytosis of marcromolecules, in exocytosis and in the intracellular transport of material between cytoplasmic compartments. Their possible role in maintaining the macromolecular composition of the plasma membrane whilst allowing recycling of excess lipid bilayer and their potential application as vehicles for the introduction of foreign macromolecules into plant cells are discussed.     

CALEB/NGC interacts with the Golgi-associated protein PIST

CALEB/NGC is a neural member of the epidermal growth factor protein family expressed in axon and synapse-rich areas of the nervous system and shown to be important for neurite formation. It can bind to the extracellular matrix proteins tenascin-R and tenascin-C. Here we show that CALEB/NGC interacts with the Golgi-associated protein PIST. PIST was originally described as an interaction partner of the small GTPase TC10 and was then found to be Golgi-associated by binding to syntaxin-6 and to be important for the transport of frizzled proteins and the cystic fibrosis transmembrane conductance regulator to the plasma membrane. In addition, PIST was demonstrated to be involved in autophagy and linked to processes of neurodegeneration. CALEB/NGC interacts with PIST in the yeast two-hybrid system. This interaction can be confirmed by co-immunoprecipitations and co-localization studies. The juxtamembrane cytoplasmic peptide segment of CALEB/NGC, highly conserved during evolution, mediates the binding to PIST. CALEB/NGC co-localizes with PIST in the Golgi apparatus of transfected COS7 cells and in Golgi-derived vesicles after brefeldin A or nocodazole treatment. Co-localization studies in primary hippocampal cells and analysis of Purkinje cells of colchicine-treated rats, serving as an in vivo model system to block microtubule-dependent transport processes, support the view that PIST is an interaction partner of CALEB/NGC and implicate that this interaction may play a role in the intracellular transport of CALEB/NGC.     

Human placental coated vesicles contain receptor-bound transferrin.

Human placental coated vesicles have been purified by a method involving sucrose-density-gradient centrifugation and treatment with wheat-germ agglutinin. These preparations were free of contamination by placental microvillus fragments. Crossed immunoelectrophoresis demonstrated that the coated vesicles contained a single serum protein, which was identified as transferrin. This transferrin was only observed after the vesicles were treated with a non-ionic detergent, and its behaviour during crossed hydrophobic-interaction immunoelectrophoresis suggested that a large proportion of it was receptor-bound. No other serum proteins, including immunoglobulin G, could be detected in these preparations. Receptor-bound transferrin was the only antigen common to placental coated vesicles and microvilli, implying that other plasma-membrane proteins are excluded from the region of membrane involved in coated-vesicle formation.     

Roles of a fimbrin and an alpha-actinin-like protein in fission yeast cell polarization and cytokinesis

Eukaryotic cells contain many actin-interacting proteins, including the alpha-actinins and the fimbrins, both of which have actin cross-linking activity in vitro. We report here the identification and characterization of both an alpha-actinin-like protein (Ain1p) and a fimbrin (Fim1p) in the fission yeast Schizosaccharomyces pombe. Ain1p localizes to the actomyosin-containing medial ring in an F-actin-dependent manner, and the Ain1p ring contracts during cytokinesis. ain1 deletion cells have no obvious defects under normal growth conditions but display severe cytokinesis defects, associated with defects in medial-ring and septum formation, under certain stress conditions. Overexpression of Ain1p also causes cytokinesis defects, and the ain1 deletion shows synthetic effects with other mutations known to affect medial-ring positioning and/or organization. Fim1p localizes both to the cortical actin patches and to the medial ring in an F-actin-dependent manner, and several lines of evidence suggest that Fim1p is involved in polarization of the actin cytoskeleton. Although a fim1 deletion strain has no detectable defect in cytokinesis, overexpression of Fim1p causes a lethal cytokinesis defect associated with a failure to form the medial ring and concentrate actin patches at the cell middle. Moreover, an ain1 fim1 double mutant has a synthetical-lethal defect in medial-ring assembly and cell division. Thus, Ain1p and Fim1p appear to have an overlapping and essential function in fission yeast cytokinesis. In addition, protein-localization and mutant-phenotype data suggest that Fim1p, but not Ain1p, plays important roles in mating and in spore formation.     

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 (homologous to E6-AP Carboxyl terminus)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.