Abstract: Caveolae appear in a multitude of processes encompassing growth regulation and trafficking. We demonstrate the abundant presence of ESA/reggie-1/flotillin-2, ATP synthase beta subunit and annexin V in endothelial caveolae by immunopurification of caveolae from vascular endothelial membrane. Five proteins are abundant in a caveolin-1 protein complex, analyzed by sucrose gradient velocity sedimentation following octyl-beta-D-glucopyranoside extraction. Caveolin-1 alpha interacts with caveolin-1beta, caveolin-2, actin, the microsomal form of NADH cytochrome B5 reductase and ESA/reggie-1/flotillin-2 as shown by co-immunoprecipitation. We propose the concept that ATP biosynthesis in caveolae regulates mechanosignaling and is induced by membrane depolarization and a proton gradient. Pressure stimuli and metabolic changes may trigger gene regulation in endothelial cells, involving a nuclear conformer of caveolin-1, shown here with an epitope-specific caveolin-1 antibody, and immediate response of ion channel activity, regulated by ESA/reggie-1/flotillin-2.
Abstract: Endothelial cell (EC) cultures of different, selected vascular beds and/or organs were screened for receptor-mediated transport of proteins with a semipermeable filter assay. In SVEC4-10 cells, a mouse lymphoid endothelial cell line, orosomucoid, albumin, insulin and LDL were transcytosed from the apical (luminal) to basal (abluminal) side by a receptor-mediated pathway. Specific LDL transcytosis involved transport of intact LDL. A pathway of degradation of LDL and basal release involved vesicles in transport to lysosomes and amino acid merocrine secretion. This newly described transcellular passage of LDL via lysosomes, as well as the standard pathway, were reduced to 70% by PEG(50)-cholesterol (PEG-Chol). Combined results of temperature-dependence analysis and PEG(50)-cholesterol sensitivity show that two pathways contribute to general LDL transcellular passage. We suggest a mechanism of domain hopping by protein membrane diffusion of receptors as the pathway for intact LDL delivery. Based on theoretical considerations we propose that active transport by protein membrane diffusion can be facilitated by an organizational structure of lipid microdomains and polar cellular organization.
Abstract: Polarized sorting of membrane proteins in epithelial cells is mediated by cytoplasmic basolateral signals or by apical signals in the transmembrane or exoplasmic domains. Basolateral signals were generally found to be dominant over apical determinants. We have generated chimeric proteins with the cytoplasmic domain of either the asialoglycoprotein receptor H1 or the transferrin receptor, two basolateral proteins, fused to the transmembrane and exoplasmic segments of aminopeptidase N, an apical protein, and analyzed them in Madin-Darby canine kidney cells. Whereas both cytoplasmic sequences induced endocytosis of the chimeras, only that of the transferrin receptor mediated basolateral expression in steady state. The H1 fusion protein, although still largely sorted to the basolateral side in biosynthetic surface transport, was subsequently resorted to the apical cell surface. We tested whether the difference in sorting between trimeric wild-type H1 and the dimeric aminopeptidase chimera was caused by the number of sorting signals presented in the oligomers. Consistent with this hypothesis, the H1 signal was fully functional in a tetrameric fusion protein with the transmembrane and exoplasmic domains of influenza neuraminidase. The results suggest that basolateral signals per se need not be dominant over apical determinants for steady-state polarity and emphasize an important contribution of the valence of signals in polarized sorting.
Abstract: Annexins form a family of proteins that are widely expressed and known to bind membranes in the presence of calcium. Two isoforms of the annexin XIII subfamily are expressed in epithelia. We previously reported that annexin XIIIb is apically localized in MDCK cells and that it is involved in raft-mediated delivery of apical proteins. We have now analyzed the properties of annexin XIIIa, which differs from annexin XIIIb by a deletion of 41 amino acids in the amino-terminal domain, and is distributed both apically and basolaterally. Annexin XIIIa binding to membranes is independent of calcium but requires its myristoyl amino-terminal modification, as observed with annexin XIIIb. Our biochemical and functional data show that annexin XIIIa behaves differently in the apical and in the basolateral compartments. Whereas annexin XIIIa apically can associate with rafts independently of calcium, the basolateral pool requires calcium for this. Annexin XIIIa, like annexin XIIIb, stimulates apical transport of influenza virus hemagglutinin but, in contrast, only annexin XIIIa inhibits basolateral transport of vesicular stomatitis virus G protein. Our results suggest that annexin XIIIa and XIIIb have specific roles in epithelial cells, and because of their structural similarities, these isoforms offer interesting tools for unravelling the functions of annexins.
Abstract: Members of the p24 family of putative cargo receptors are proposed to contain retrograde and anterograde trafficking signals in their cytoplasmic domain to facilitate coat protein binding and cycling in the secretory pathway. We have analyzed the role of the transmembrane domain (TMD) of a p24 protein isolated from COPI-coated intra-Golgi transport vesicles. CD8-p24 chimeras were transiently expressed in COS7 cells and analyzed by immunofluorescence and pulse-chase experiments. The localization and transit of the wild-type chimera from the endoplasmic reticulum (ER) through the Golgi complex involved a glutamic acid residue and a conserved glutamine in the TMD. The TMD glutamic acid mediated the localization of the chimeras to the ER in the absence of the conserved glutamine. Efficient ER exit required the TMD glutamine and was further facilitated by a pair of phenylalanine residues in the cytoplasmic tail. TMD residues of p24 proteins may mediate the interaction with integral membrane proteins of the vesicle budding machinery to ensure p24 packaging into transport vesicles.
Abstract: In polarized MDCK cells, proteins and lipids are sorted in the trans-Golgi network /TGN) and packaged into different vesicular carriers that are delivered to the apical or basolateral cell surface. To gain insight into the sorting and trafficking machinery, we have previously isolated TGN-derived carrier vesicles from perforated MDCK cells. The composition of immuno-isolated apical and basolateral carriers was mapped by two-dimensional (2-D) gel electrophoresis. Here we describe the identification of several components of the vesicle fraction by using three different methods. 2-D gel comigration was performed with carrier vesicles isolated from metabolically labeled MDCK cells and human epidermal keratinocyte lysates. This allowed us to assign eleven known components by a comparison with the comprehensive keratinocyte 2-D gel database. These comprised two members of the 14-3-3 family of proteins that have been implicated in vesicular trafficking. Five proteins were purified from preparative 2-D gels and identified by peptide microsequencing, including the beta1 and beta2 subunit of trimeric G proteins and an annexin II variant. A member of the SNARE family of proteins was identified by immunoblotting. The combination of 2-D gel electrophoresis and 2-D gel databases allows the rapid assessment of the purity of subcellular fractions and to characterize components involved in vesicular transport.
Abstract: Cytoplasmic domains of members of the p24 family of putative cargo receptors were shown to bind to coatomer, the coat protein of COPI-coated transport vesicles. Domains that contained dilysine endoplasmic reticulum retrieval signals bound the alpha-, beta'-, and epsilon-COP subunits of coatomer, whereas other p24 domains bound the beta-, gamma-, and zeta-COP subunits and required a phenylalanine-containing motif. Transit of a CD8-p24 chimera from the endoplasmic reticulum through the Golgi complex was slowed when the phenylalanine motif was mutated, suggesting that this motif may function as an anterograde transport signal. The either-or bimodal binding of coatomer to p24 tails suggests models for how coatomer can potentially package retrograde-directed and anterograde-directed cargo into distinct COPI-coated vesicles.
Abstract: VIP36 was isolated from MDCK cells as a component of glycolipid-enriched detergent-insoluble complexes. The protein is localized to the Golgi apparatus and the cell surface, and belongs to a new family of legume lectin homologues in the animal secretory pathway that might be involved in the trafficking of glycoproteins, glycolipids or both. Here we show that VIP36 is N-glycosylated and expressed in organs abundant in epithelial cells as well as in non-epithelial organs. Our studies demonstrate that the recombinant exoplasmic/luminal domain of VIP36 binds Ca2+ and that the protein decorates internal membrane structures of MDCK cells in vitro that are distinct from the Golgi apparatus. This binding requires Ca2+ and can be specifically inhibited by N-acetyl-D-galactosamine. The recombinant protein was used for affinity chromatography. Glycopeptides obtained from [3H]galactose-labelled cells bind to VIP36 and can be eluted with N-acetyl-D-galactosamine. Our data imply that VIP36 functions as a lectin in post-Golgi trafficking.
Abstract: Formation of non-clathrin-coated vesicles requires the recruitment of several cytosolic factors to the Golgi membrane. To identify membrane proteins involved in this budding process, a highly abundant type I transmembrane protein (p23) was isolated from mammalian Golgi-derived COPI-coated vesicles, and its cDNA was cloned and sequenced. It belongs to the p24 family of proteins involved in the budding of transport vesicles (Stamnes, M.A., M.W. Craighead, M.H. Hoe, N. Lampen, S. Geromanos, P. Tempst, and J.E. Rothman. 1995. Proc. Natl. Acad. Sci. USA. 92:8011-8015). p23 consists of a large NH2-terminal luminal domain and a short COOH-terminal cytoplasmic tail (-LRRFFKAKKLIE-CO2-) that shows similarity, but not identity, with the sequence motif-KKXX-CO2-, known as a signal for retrieval of escaped ER-resident membrane proteins (Jackson, M.R., T. Nilsson, and P.A. Peterson. 1990. EMBO (Eur. Mol. Biol. Organ.) J. 9:3153-3162; Nilsson, T., M. Jackson, and P.A. Peterson. 1989. Cell. 58:707-718). The cytoplasmic tail of p23 binds to coatomer with similar efficiency as known KKXX motifs. However, the p23 tail differs from the KKXX motif in having an additional motif needed for binding of coatomer. p23 is localized to Golgi cisternae and, during vesicle formation, it concentrates into COPI-coated buds and vesicles. Biochemical analysis revealed that p23 is enriched in vesicles by a factor of approximately 20, as compared with the donor Golgi fraction, and is present in amounts stoichiometric to the small GTP-binding protein ADP-ribosylation factor (ARF) and coatomer. From these data we conclude that p23 represents a Golgi-specific receptor for coatomer involved in the formation of COPI-coated vesicles.
Abstract: The sorting of apical and basolateral proteins into vesicular carriers takes place in the trans-Golgi network (TGN) in MDCK cells. We have previously analyzed the protein composition of immunoisolated apical and basolateral transport vesicles and have now identified a component that is highly enriched in apical vesicles. Isolation of the encoding cDNA revealed that this protein, annexin XIIIb, is a new isoform of the epithelial specific annexin XIII sub-family which includes the previously described intestine-specific annexin (annexin XIIIa; Wice, B. M., and J. I. Gordon. 1992. J. Cell Biol. 116:405-422). Annexin XIIIb differs from annexin XIIIa in that it contains a unique insert of 41 amino acids in the NH2 terminus and is exclusively expressed in dog intestine and kidney. Immunofluorescence microscopy demonstrated that annexin XIIIb was localized to the apical plasma membrane and underlying punctate structures. Since annexins have been suggested to play a role in membrane-membrane interactions in exocytosis and endocytosis, we investigated whether annexin XIIIb is involved in delivery to the apical cell surface. To this aim we used permeabilized MDCK cells and a cytosol-dependent in vitro transport assay. Antibodies specific for annexin XIIIb significantly inhibited the transport of influenza virus hemagglutinin from the TGN to the apical plasma membrane while the transport of vesicular stomatitis virus glycoprotein to the basolateral cell surface was unaffected. We propose that annexin XIIIb plays a role in vesicular transport to the apical plasma membrane in MDCK cells.
Abstract: VIP17 is a proteolipid enriched in the CHAPS-insoluble complexes from MDCK cells, and a candidate component of the molecular machinery responsible for the sorting and targeting of proteins to the apical surface. Cloning and sequencing of the cDNA encoding the protein revealed that it is the canine homolog of the human and rat MAL proteins. Analysis by immunofluorescence microscopy of epitope-tagged VIP17/MAL expressed transiently in BHK cells and stably in MDCK cells revealed a perinuclear, vesicular, and plasmalemmal staining. In MDCK cells the distribution was mainly in vesicular structures in the apical cytoplasm. These and other results suggest that VIP17/MAL is an important component in vesicular trafficking cycling between the Golgi complex and the apical plasma membrane.
Abstract: The remarkable quantities of myelin membrane produced by oligodendrocytes has led us to examine the mechanisms involved in the sorting and transport of proteins and lipids during myelinogenesis. Noting that it has been proposed that proteins destined for the apical surface of polarized epithelial cells co-cluster with glycolipid-rich microdomains during sorting and transport from the trans-Golgi network (Simons and van Meer: Biochemistry 27:6197-6202, 1988; Simons and Wandinger-Ness: Cell 62:207-210, 1990), we hypothesized that the glycolipid-rich oligodendrocytes may adopt this mechanism for myelinogenesis. Protein-lipid complexes from oligodendrocytes and myelin were isolated utilizing detergent insolubility and two-dimensional gel electrophoresis. A developmentally regulated protein, MVP17 (myelin vesicular protein of 17 kDa), was identified. Microsequencing of the N-terminal peptide revealed a high homology to human T-cell MAL protein (Alonso and Weissman: Proc Natl Acad Sci USA 84:1997-2001, 1987). The corresponding MVP17 cDNA was isolated from an oligodendrocyte cDNA library. The predicted protein sequence showed 88.9% identity with MAL, and the hydrophobicity profile suggested four transmembrane domains. In vitro translation demonstrated a signal at the deduced Mr of approximately 17 kDa. Northern analyses indicated that MVP17 mRNA expression is restricted to brain and kidney and that this expression is up-regulated in oligodendrocytes and brain during the period of active myelination. These data suggest that MVP17 is involved in myelin biogenesis and/or myelin function.
Abstract: Prohibitin is a ubiquitously expressed protein with antiproliferative properties. When rat prohibitin tagged with a carboxy-terminal c-Myc epitope was expressed in baby hamster kidney cells the protein was targeted to mitochondria. In immunofluorescence microscopy prohibitin colocalized with a mitochondrial marker E3. Immunoelectron microscopy revealed that prohibitin was associated with the periphery of mitochondria. The amino-terminus of prohibitin shares characteristics of the known mitochondrial import signals, and positioning of the tag at the N-terminus causes accumulation of the protein in the cytoplasm. These findings help to direct functional studies on prohibitin and suggest that a mitochondrial protein may act as a tumor suppressor.
Abstract: In simple epithelial cells, apical and basolateral proteins and lipids in transit to the cell surface are sorted in the trans-Golgi network. We have recently isolated detergent-insoluble complexes from Madin-Darby canine kidney cells that are enriched in glycosphingolipids, apical cargo and a subset of the proteins of the exocytic carrier vesicles. The vesicular proteins are thought to be involved in protein sorting and include VIP21-caveolin. The vesicular protein VIP36 (36 kDa vesicular integral membrane protein) has been purified from a CHAPS-insoluble residue and a cDNA encoding VIP36 has been isolated. The N-terminal 31 kDa luminal/exoplasmic domain of the encoded protein shows homology to leguminous plant lectins. The transiently expressed protein is localized to the Golgi apparatus, endosomal and vesicular structures and the plasma membrane, as predicted for a protein involved in transport between the Golgi and the cell surface. It is diffusely localized on the plasma membrane but can be redistributed by antibody modulation into caveolae and clathrin-coated pits. We speculate that VIP36 binds to sugar residues of glycosphingolipids and/or glycosylphosphatidyl-inositol anchors and might provide a link between the extracellular/luminal face of glycolipid rafts and the cytoplasmic protein segregation machinery.
Abstract: In simple epithelial cells, the delivery of apical and basolateral proteins to the cell surface is mediated by sorting in the trans-Golgi network and transport via separate vesicular carriers. In order to identify the molecular machinery involved in protein sorting, we have recently studied a detergent-insoluble complex in Madin-Darby canine kidney (MDCK) cells, following CHAPS extraction of exocytic carrier vesicles, specifically including the apical marker protein influenza hemagglutinin (HA). Previously, a Triton X-100 insoluble membrane residue that was enriched in glycosylphosphatidylinositol-anchored (GPI) proteins and glycolipids was characterized and implicated in transport to the apical cell surface [Brown, D., & Rose, J. (1991) Cell 68, 533-544]. In this report, the protein compositions of the CHAPS and Triton complexes have been compared by two-dimensional gel analysis. Only a few major membrane proteins are found in the complexes. The protein compositions are qualitatively similar, but differ quantitatively in the individual components. The CHAPS complex is depleted of GPI-linked proteins and retains a minor fraction of lipids similar in composition to that of the Triton X-100 insoluble complex. We propose that in vivo the complexes form part of a sorting platform that mediates protein segregation and delivery to the apical cell surface.
Abstract: Uncleaved signal-anchor sequences of membrane proteins inserted into the endoplasmic reticulum initiate the translocation of either the amino-terminal or the carboxyl-terminal polypeptide segment across the bilayer. Which topology is acquired is not determined by the apolar segment of the signal but rather by the hydrophilic sequences flanking it. To study the role of charged residues in determining the membrane topology, the insertion of mutants of the asialoglycoprotein receptor H1, a single-spanning protein with a cytoplasmic amino terminus, was analyzed in transfected COS-7 cells. When the charged amino acids flanking the hydrophobic signal were mutated to residues of opposite charge, half the polypeptides inserted with the inverted orientation. When, in addition, the amino-terminal domain of the mutant protein was truncated, approximately 90% of the polypeptides acquired the inverted topology. The transmembrane orientation appears to be primarily determined by the charges flanking the signal sequence but is modulated by the domains to be translocated.
