Isolation of a brefeldin A-inhibited guanine nucleotide-exchange protein for ADP ribosylation factor (ARF) 1 and ARF3 that contains a Sec7-like domain

Brefeldin A (BFA) inhibited the exchange of ADP ribosylation factor (ARF)-bound GDP for GTP by a Golgi-associated guanine nucleotide-exchange protein (GEP) [Helms, J. B. & Rothman, J. E. (1992) Nature (London) 360, 352–354; Donaldson, J. G., Finazzi, D. & Klausner, R. D. (1992) Nature (London) 360, 350–352]. Cytosolic ARF GEP was also inhibited by BFA, but after purification from bovine brain and rat spleen, it was no longer BFA-sensitive [Tsai, S.-C., Adamik, R., Moss, J. & Vaughan, M. (1996) Proc. Natl. Acad. Sci. USA 93, 305–309]. We describe here purification from bovine brain cytosol of a BFA-inhibited GEP. After chromatography on DEAE–Sephacel, hydroxylapatite, and Mono Q and precipitation at pH 5.8, GEP was eluted from Superose 6 as a large molecular weight complex at the position of thyroglobulin (≈670 kDa). After SDS/PAGE of samples from column fractions, silver-stained protein bands of ≈190 and 200 kDa correlated with activity. BFA-inhibited GEP activity of the 200-kDa protein was demonstrated following electroelution from the gel and renaturation by dialysis. Four tryptic peptides from the 200-kDa protein had amino acid sequences that were 47% identical to sequences in Sec7 from Saccharomyces cerevisiae (total of 51 amino acids), consistent with the view that the BFA-sensitive 200-kDa protein may be a mammalian counterpart of Sec7 that plays a similar role in cellular vesicular transport and Sec7 may be a GEP for one or more yeast ARFs.

Interaction of measles virus glycoproteins with the surface of uninfected peripheral blood lymphocytes induces immunosuppression in vitro

A marked suppression of immune function has long been recognized as a major cause of the high morbidity and mortality rate associated with acute measles. As a hallmark of measles virus (MV)-induced immunosuppression, peripheral blood lymphocytes (PBLs) isolated from patients exhibit a significantly reduced capacity to proliferate in response to mitogens, allogens, or recall antigens. In an in vitro system we show that proliferation of naive PBLs [responder cells (RCs)] in response to a variety of stimuli was significantly impaired after cocultivation with MV-infected, UV-irradiated autologous PBLs [presenter cells (PCs)]. We further observed that a 50% reduction in proliferation of RCs could still be observed when the ratio of PC to RC was 1:100. The effect was completely abolished after physical separation of the two populations, which suggests that soluble factors were not involved. Proliferative inhibition of the RCs was observed after short cocultivation with MV-infected cells, which indicates that surface contact between one or more viral proteins and the RC population was required. We identified that the complex of both MV glycoproteins, F and H, is critically involved in triggering MV-induced suppression of mitogen-dependent proliferation, since the effect was not observed (i) using a recombinant MV in which F and H were replaced with vesicular stomatitis virus G or (ii) when either of these proteins was expressed alone. Coexpression of F and H, however, lead to a significant proliferative inhibition in the RC population. Our data indicate that a small number of MV-infected PBLs can induce a general nonresponsiveness in uninfected PBLs by surface contact, which may, in turn, account for the general suppression of immune responses observed in patients with acute measles.

Cloning and expression of a cDNA encoding a bovine brain brefeldin A-sensitive guanine nucleotide-exchange protein for ADP-ribosylation factor

A 200-kDa guanine nucleotide-exchange protein (p200 or GEP) for ADP-ribosylation factors 1 and 3 (ARF1 and ARF3) that was inhibited by brefeldin A (BFA) was purified earlier from cytosol of bovine brain cortex. Amino acid sequences of four tryptic peptides were 47% identical to that of Sec7 from Saccharomyces cerevisiae, which is involved in vesicular trafficking in the Golgi. By using a PCR-based procedure with two degenerate primers representing sequences of these peptides, a product similar in size to Sec7 that contained the peptide sequences was generated. Two oligonucleotides based on this product were used to screen a bovine brain library, which yielded one clone that was a partial cDNA for p200. The remainder of the cDNA was obtained by 5′ and 3′ rapid amplification of cDNA ends (RACE). The ORF of the cDNA encodes a protein of 1,849 amino acids (≈208 kDa) that is 33% identical to yeast Sec7 and 50% identical in the Sec7 domain region. On Northern blot analysis of bovine tissues, a ≈7.4-kb mRNA was identified that hybridized with a p200 probe; it was abundant in kidney, somewhat less abundant in lung, spleen, and brain, and still less abundant in heart. A six-His-tagged fusion protein synthesized in baculovirus-infected Sf9 cells demonstrated BFA-inhibited GEP activity, confirming that BFA sensitivity is an intrinsic property of this ARF GEP and not conferred by another protein component of the complex from which p200 was originally purified.

Basolateral membrane targeting of a renal-epithelial inwardly rectifying potassium channel from the cortical collecting duct, CCD-IRK3, in MDCK cells

We recently cloned an inward-rectifying K channel (Kir) cDNA, CCD-IRK3 (mKir 2.3), from a cortical collecting duct (CCD) cell line. Although this recombinant channel shares many functional properties with the “small-conductance” basolateral membrane Kir channel in the CCD, its precise subcellular localization has been difficult to elucidate by conventional immunocytochemistry. To circumvent this problem, we studied the targeting of several different epitope-tagged CCD-IRK3 in a polarized renal epithelial cell line. Either the 11-amino acid span of the vesicular stomatitis virus (VSV) G glycoprotein (P5D4 epitope) or a 6-amino acid epitope of the bovine papilloma virus capsid protein (AU1) was genetically engineered on the extreme N terminus of CCD-IRK3. As determined by patch-clamp and two-microelectrode voltage-clamp analyses in Xenopus oocytes, neither tag affected channel function; no differences in cation selectivity, barium block, single channel conductance, or open probability could be distinguished between the wild-type and the tagged constructs. MDCK cells were transfected with tagged CCD-IRK3, and several stable clonal cell lines were generated by neomycin-resistance selection. Immunoprecipitation studies with anti-P5D4 or anti-AU1 antibodies readily detected the predicted-size 50-kDa protein in the transfected cells lines but not in wild-type or vector-only (PcB6) transfected MDCK cells. As visualized by indirect immunofluorescence and confocal microscopy, both the tagged CCD-IRK3 forms were exclusively detected on the basolateral membrane. To assure that the VSV G tag was not responsible for the targeting, the P5D4 epitope modified by a site-directed mutagenesis (Y2F) to remove a potential basolateral targeting signal contained in this tag. VSV(Y2F) was also detected exclusively on the basolateral membrane, confirming bona fide IRK3 basolateral expression. These observations, with our functional studies, suggest that CCD-IRK3 may encode the small-conductance CCD basolateral K channel.

A widely expressed βIII spectrin associated with Golgi and cytoplasmic vesicles

Spectrin is an important structural component of the plasma membrane skeleton. Heretofore-unidentified isoforms of spectrin also associate with Golgi and other organelles. We have discovered another member of the β-spectrin gene family by homology searches of the GenBank databases and by 5′ rapid amplification of cDNA ends of human brain cDNAs. Collectively, 7,938 nucleotides of contiguous clones are predicted to encode a 271,294-Da protein, called βIII spectrin, with conserved actin-, protein 4.1-, and ankyrin-binding domains, membrane association domains 1 and 2, a spectrin dimer self-association site, and a pleckstrin-homology domain. βIII spectrin transcripts are concentrated in the brain and present in the kidneys, liver, and testes and the prostate, pituitary, adrenal, and salivary glands. All of the tested tissues contain major 9.0-kb and minor 11.3-kb transcripts. The human βIII spectrin gene (SPTBN2) maps to chromosome 11q13 and the mouse gene (Spnb3) maps to a syntenic region close to the centromere on chromosome 19. Indirect immunofluorescence studies of cultured cells using antisera specific to human βIII spectrin reveal a Golgi-associated and punctate cytoplasmic vesicle-like distribution, suggesting that βIII spectrin associates with intracellular organelles. This distribution overlaps that of several Golgi and vesicle markers, including mannosidase II, p58, trans-Golgi network (TGN)38, and β-COP and is distinct from the endoplasmic reticulum markers calnexin and Bip. Liver Golgi membranes and other vesicular compartment markers cosediment in vitro with βIII spectrin. βIII spectrin thus constitutes a major component of the Golgi and vesicular membrane skeletons.

Network formation of lipid membranes: Triggering structural transitions by chain melting

Phospholipids when dispersed in excess water generally form vesicular membrane structures. Cryo-transmission and freeze-fracture electron microscopy are combined here with calorimetry and viscometry to demonstrate the reversible conversion of phosphatidylglycerol aqueous vesicle suspensions to a three-dimensional structure that consists of extended bilayer networks. Thermodynamic analysis indicates that the structural transitions arise from two effects: (i) the enhanced membrane elasticity accompanying the lipid state fluctuations on chain melting and (ii) solvent-associated interactions (including electrostatics) that favor a change in membrane curvature. The material properties of the hydrogels and their reversible formation offer the possibility of future applications, for example in drug delivery, the design of structural switches, or for understanding vesicle fusion or fission processes.

Peroxynitrite rapidly permeates phospholipid membranes

Peroxynitrite (ONOO−) is a potent oxidant implicated in a number of pathophysiological processes. The activity of ONOO− is related to its accessibility to biological targets before its spontaneous decomposition (t1/2 ≈ 1 s at pH 7.4, 37°C). Using model phospholipid vesicular systems and manganese porphyrins as reporter molecules, we demonstrated that ONOO− freely crosses phospholipid membranes. The calculated permeability coefficient for ONOO− is ≈8.0 × 10−4 cm⋅s−1, which compares well with that of water and is ≈400 times greater than that of superoxide. We suggest that ONOO− is a significant biological effector molecule not only because of its reactivity but also because of its high diffusibility.

Protein kinase A activity is required for the budding of constitutive transport vesicles from the trans-Golgi network

We have examined the role played by protein kinase A (PKA) in vesicle-mediated protein transport from the trans-Golgi network (TGN) to the cell surface. In vivo this transport step was inhibited by inhibitors of PKA catalytic subunits (C-PKA) such as the compound known as H89 and a myristoylated form of the inhibitory peptide sequence contained in the thermostable PKA inhibitor. Inhibition by H89 occurred at an early stage during the transfer of vesicular stomatitis virus G glycoprotein from the TGN to the cell surface. Reversal from this inhibition correlated with a transient increase in the number of free coated vesicles in the Golgi area. Vesicle budding from the TGN was studied in vitro using vesicular stomatitis virus-infected, permeabilized cells. Addition to this assay of C-PKA stimulated vesicle release while it was suppressed by PKA inhibitory peptide, H89, and antibody against C-PKA. Furthermore, vesicle release was decreased when PKA-depleted cytosol was used and restored by addition of C-PKA. These results indicate a regulatory role for PKA activity in the production of constitutive transport vesicles from the TGN.

A system for functional analysis of Ebola virus glycoprotein

Ebola virus causes hemorrhagic fever in humans and nonhuman primates, resulting in mortality rates of up to 90%. Studies of this virus have been hampered by its extraordinary pathogenicity, which requires biosafety level 4 containment. To circumvent this problem, we developed a novel complementation system for functional analysis of Ebola virus glycoproteins. It relies on a recombinant vesicular stomatitis virus (VSV) that contains the green fluorescent protein gene instead of the receptor-binding G protein gene (VSVΔG*). Herein we show that Ebola Reston virus glycoprotein (ResGP) is efficiently incorporated into VSV particles. This recombinant VSV with integrated ResGP (VSVΔG*-ResGP) infected primate cells more efficiently than any of the other mammalian or avian cells examined, in a manner consistent with the host range tropism of Ebola virus, whereas VSVΔG* complemented with VSV G protein (VSVΔG*-G) efficiently infected the majority of the cells tested. We also tested the utility of this system for investigating the cellular receptors for Ebola virus. Chemical modification of cells to alter their surface proteins markedly reduced their susceptibility to VSVΔG*-ResGP but not to VSVΔG*-G. These findings suggest that cell surface glycoproteins with N-linked oligosaccharide chains contribute to the entry of Ebola viruses, presumably acting as a specific receptor and/or cofactor for virus entry. Thus, our VSV system should be useful for investigating the functions of glycoproteins from highly pathogenic viruses or those incapable of being cultured in vitro.

Association of Rab25 and Rab11a with the Apical Recycling System of Polarized Madin–Darby Canine Kidney Cells

Recent evidence suggests that apical and basolateral endocytic pathways in epithelia converge in an apically located, pericentriolar endosomal compartment termed the apical recycling endosome. In this compartment, apically and basolaterally internalized membrane constituents are thought to be sorted for recycling back to their site of origin or for transcytosis to the opposite plasma membrane domain. We report here that in the epithelial cell line Madin–Darby Canine Kidney (MDCK), antibodies to Rab11a label an apical pericentriolar endosomal compartment that is dependent on intact microtubules for its integrity. Furthermore, this compartment is accessible to a membrane-bound marker (dimeric immunoglobulin A [IgA]) internalized from either the apical or basolateral pole, functionally defining it as the apical recycling endosome. We have also examined the role of a closely related epithelial-specific Rab, Rab25, in the regulation of membrane recycling and transcytosis in MDCK cells. When cDNA encoding Rab25 was transfected into MDCK cells, the protein colocalized with Rab11a in subapical vesicles. Rab25 transfection also altered the distribution of Rab11a, causing the coalescence of immunoreactivity into multiple denser vesicular structures not associated with the centrosome. Nevertheless, nocodazole still dispersed these vesicles, and dimeric IgA internalized from either the apical or basolateral membrane was detected in endosomes labeled with antibodies to both Rab11a and Rab25. Overexpression of Rab25 decreased the rate of IgA transcytosis and of apical, but not basolateral, recycling of internalized ligand. Conversely, expression of the dominant-negative Rab25T26N did not alter either apical recycling or transcytosis. These results indicate that both Rab11a and Rab25 associate with the apical recycling system of epithelial cells and suggest that Rab25 may selectively regulate the apical recycling and/or transcytotic pathways.

Structural and Functional Analysis of a Novel Coiled-Coil Protein Involved in Ypt6 GTPase-regulated Protein Transport in Yeast

The yeast transport GTPase Ypt6p is dispensable for cell growth and secretion, but its lack results in temperature sensitivity and missorting of vacuolar carboxypeptidase Y. We previously identified four yeast genes (SYS1, 2, 3, and 5) that on high expression suppressed these phenotypic alterations. SYS3 encodes a 105-kDa protein with a predicted high α-helical content. It is related to a variety of mammalian Golgi-associated proteins and to the yeast Uso1p, an essential protein involved in docking of endoplasmic reticulum–derived vesicles to the cis-Golgi. Like Uso1p, Sys3p is predominatly cytosolic. According to gel chromatographic, two-hybrid, and chemical cross-linking analyses, Sys3p forms dimers and larger protein complexes. Its loss of function results in partial missorting of carboxypeptidase Y. Double disruptions of SYS3 and YPT6 lead to a significant growth inhibition of the mutant cells, to a massive accumulation of 40- to 50-nm vesicles, to an aggravation of vacuolar protein missorting, and to a defect in α-pheromone processing apparently attributable to a perturbation of protease Kex2p cycling between the Golgi and a post-Golgi compartment. The results of this study suggest that Sys3p, like Ypt6p, acts in vesicular transport (presumably at a vesicle-docking stage) between an endosomal compartment and the most distal Golgi compartment.

GS32, a Novel Golgi SNARE of 32 kDa, Interacts Preferentially with Syntaxin 6

Syntaxin 1, synaptobrevins or vesicle-associated membrane proteins, and the synaptosome-associated protein of 25 kDa (SNAP-25) are key molecules involved in the docking and fusion of synaptic vesicles with the presynaptic membrane. We report here the molecular, cell biological, and biochemical characterization of a 32-kDa protein homologous to both SNAP-25 (20% amino acid sequence identity) and the recently identified SNAP-23 (19% amino acid sequence identity). Northern blot analysis shows that the mRNA for this protein is widely expressed. Polyclonal antibodies against this protein detect a 32-kDa protein present in both cytosol and membrane fractions. The membrane-bound form of this protein is revealed to be primarily localized to the Golgi apparatus by indirect immunofluorescence microscopy, a finding that is further established by electron microscopy immunogold labeling showing that this protein is present in tubular-vesicular structures of the Golgi apparatus. Biochemical characterizations establish that this protein behaves like a SNAP receptor and is thus named Golgi SNARE of 32 kDa (GS32). GS32 in the Golgi extract is preferentially retained by the immobilized GST–syntaxin 6 fusion protein. The coimmunoprecipitation of syntaxin 6 but not syntaxin 5 or GS28 from the Golgi extract by antibodies against GS32 further sustains the preferential interaction of GS32 with Golgi syntaxin 6.

Differential Epitope Tagging of Actin in Transformed Drosophila Produces Distinct Effects on Myofibril Assembly and Function of the Indirect Flight Muscle

We have tested the impact of tags on the structure and function of indirect flight muscle (IFM)-specific Act88F actin by transforming mutant Drosophila melanogaster, which do not express endogenous actin in their IFMs, with tagged Act88F constructs. Epitope tagging is often the method of choice to monitor the fate of a protein when a specific antibody is not available. Studies addressing the functional significance of the closely related actin isoforms rely almost exclusively on tagged exogenous actin, because only few antibodies exist that can discriminate between isoforms. Thereby it is widely presumed that the tag does not significantly interfere with protein function. However, in most studies the tagged actin is expressed in a background of endogenous actin and, as a rule, represents only a minor fraction of the total actin. The Act88F gene encodes the only Drosophila actin isoform exclusively expressed in the highly ordered IFM. Null mutations in this gene do not affect viability, but phenotypic effects in transformants can be directly attributed to the transgene. Transgenic flies that express Act88F with either a 6x histidine tag or an 11-residue peptide derived from vesicular stomatitis virus G protein at the C terminus were flightless. Overall, the ultrastructure of the IFM resembled that of the Act88F null mutant, and only low amounts of C-terminally tagged actins were found. In contrast, expression of N-terminally tagged Act88F at amounts comparable with that of wild-type flies yielded fairly normal-looking myofibrils and partially reconstituted flight ability in the transformants. Our findings suggest that the N terminus of actin is less sensitive to modifications than the C terminus, because it can be tagged and still polymerize into functional thin filaments.

Phototactic Migration of Dictyostelium Cells Is Linked to a New Type of Gelsolin-related Protein

The molecular and functional characterization of a 125-kDa Ca2+-extractable protein of the Triton X-100–insoluble fraction of Dictyostelium cells identified a new type of a gelsolin-related molecule. In addition to its five gelsolin segments, this gelsolin-related protein of 125 kDa (GRP125) reveals a number of unique domains, two of which are predicted to form coiled-coil regions. Another distinct attribute of GRP125 concerns the lack of sequence elements known to be essential for characteristic activities of gelsolin-like proteins, i.e. the severing, capping, or nucleation of actin filaments. The subcellular distribution of GRP125 to vesicular compartments suggests an activity of GRP125 different from actin-binding, gelsolin-related proteins. GRP125 expression is tightly regulated and peaks at the transition to the multicellular pseudoplasmodial stage of Dictyostelium development. GRP125 was found indispensable for slug phototaxis, because slugs fail to correctly readjust their orientation in the absence of GRP125. Analysis of the GRP125-deficient mutant showed that GRP125 is required for coupling photodetection to the locomotory machinery of slugs. We propose that GRP125 is essential in the natural environment for the propagation of Dictyostelium spores. We also present evidence for further representatives of the GRP125 type in Dictyostelium, as well as in heterologous cells from lower to higher eukaryotes.

Morphological and Functional Association of Sec22b/ERS-24 with the pre-Golgi Intermediate Compartment

Yeast Sec22p participates in both anterograde and retrograde vesicular transport between the endoplasmic reticulum (ER) and the Golgi apparatus by functioning as a v-SNARE (soluble N-ethylmaleimide-sensitive factor [NSF] attachment protein receptor) of transport vesicles. Three mammalian proteins homologous to Sec22p have been identified and are referred to as Sec22a, Sec22b/ERS-24, and Sec22c, respectively. The existence of three homologous proteins in mammalian cells calls for detailed cell biological and functional examinations of each individual protein. The epitope-tagged forms of all three proteins have been shown to be primarily associated with the ER, although functional examination has not been carefully performed for any one of them. In this study, using antibodies specific for Sec22b/ERS-24, it is revealed that endogenous Sec22b/ERS-24 is associated with vesicular structures in both the perinuclear Golgi and peripheral regions. Colabeling experiments for Sec22b/ERS-24 with Golgi mannosidase II, the KDEL receptor, and the envelope glycoprotein G (VSVG) of vesicular stomatitis virus (VSV) en route from the ER to the Golgi under normal, brefeldin A, or nocodazole-treated cells suggest that Sec22b/ERS-24 is enriched in the pre-Golgi intermediate compartment (IC). In a well-established semi-intact cell system that reconstitutes transport from the ER to the Golgi, transport of VSVG is inhibited by antibodies against Sec22b/ERS-24. EGTA is known to inhibit ER–Golgi transport at a stage after vesicle/transport intermediate docking but before the actual fusion event. Antibodies against Sec22b/ERS-24 inhibit ER–Golgi transport only when they are added before the EGTA-sensitive stage. Transport of VSVG accumulated in pre-Golgi IC by incubation at 15°C is also inhibited by Sec22b/ERS-24 antibodies. Morphologically, VSVG is transported from the ER to the Golgi apparatus via vesicular intermediates that scatter in the peripheral as well as the Golgi regions. In the presence of antibodies against Sec22b/ERS-24, VSVG is seen to accumulate in these intermediates, suggesting that Sec22b/ERS-24 functions at the level of the IC in ER–Golgi transport.