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PubMed1995

The interaction of coatomer with inositol polyphosphates is conserved in Saccharomyces cerevisiae.

Coatomer is an oligomeric complex of coat proteins that regulates vesicular traffic through the Golgi complex and from the Golgi to the endoplasmic reticulum [Pelham (1994) Cell 79, 1125-1127]. We have investigated whether the binding of InsP6 to mammalian coatomer [Fleischer, Xie, Mayrleitner, Shears and Fleischer (1994) J. Biol. Chem. 269, 17826-17832] is conserved in the genetically amenable model Saccharomyces cerevisiae. We have isolated coatomer from S. cerevisiae and found it to bind InsP6 at two apparent classes of binding sites (KD1 = 0.8 +/- 0.2 nM; KD2 = 361 +/- 102 nM). Ligand specificity was studied by displacing 4.5 nM [3H]InsP6 from coatomer with various Ins derivatives. The following IC50 values (nM) were obtained: myo-InsP6 = 6; bis(diphospho)inositol tetrakisphosphate = 6; diphosphoinositol pentakisphosphate = 6; scyllo-InsP6 = 12; Ins(1,3,4,5,6)P5 = 13; Ins(1,2,4,5,6)P5 = 22; Ins(1,3,4,5)P4 = 22; 1-O-(1,2-di-O-octanoyl-sn-glycero-3-phospho)-D-Ins(3,4,5)P3 = 290. Less than 10% of the 3H label was displaced by 1 microM of either Ins(1,4,5)P3 or inositol hexakis-sulphate. A cell-free lysate of S. cerevisiae synthesized diphosphoinositol polyphosphates (PP-InsPn) from InsP6, but our binding data, plus measurements of the relative levels of inositol polyphosphates in intact yeast [Hawkins, Stephens and Piggott (1993) J. Biol. Chem. 268, 3374-3383], indicate that InsP6 is the major physiologically relevant ligand. Thus a reconstituted vesicle trafficking system using coatomer and other functionally related components isolated from yeast should be a useful model for elucidating the functional significance of the binding of InsP6 by coatomer.

Binding Sites↗
PubMed1986

Anti-sense regions in satellite RNA of cucumber mosaic virus form stable complexes with the viral coat protein gene.

The interaction in vitro of the RNA of the Q-strain of cucumber mosaic virus (CMV) with its satellite RNA (sat-RNA) has been studied. In hybridisation reactions containing 30% formamide at 45 degrees, sat-RNA binds to CMV RNA 3 and 4 but not to CMV RNA 1 and 2 or RNA from tobacco mosaic virus and alfalfa mosaic virus. The viral coat protein gene present in RNA 3 and 4 contains the site of binding but this region does not contain complementary sequences of any significant length to the sat-RNA sequence. However, the optimum alignment of short complementary sequences present in these regions revealed a stable structure in which it is proposed that sat-RNA twists around the coat protein gene so that two separate blocks of nucleotides in sat-RNA base pair in opposite directions with two adjacent blocks in the coat protein gene to form a knot-like structure. The binding site is a region of 33 nucleotides within the coding region of the coat protein gene which base pairs with residues 98-113 and 134-152 of sat-RNA. The possibility of the binding region of sat-RNA functioning as an "anti-sense" sequence in regulation of the viral coat protein synthesis is discussed.

Base Sequence↗
PubMed1976

Inhibition by zinc of rhinovirus protein cleavage: interaction of zinc with capsid polypeptides.

Zinic ions rapidly inhibit virus production in HeLa cells infected with human rhinovirus type 1A and lead to the accumulation of human rhinovirus type 1A precursor polypeptides. The degree to which cleavage of these precursors is inhibited is directly dependent on the quantity of cell-associated zinc. Proteolysis resumes after the removal of zinc-containing medium, and the accumulated viral precursors are cleaved predominantly to stable virus polypeptides. The precursors stabilized at the lowest zinc levels are those that contain capsid protein sequences. Furthermore, added zinc is bound to human rhinovirus type 1A capsids and prevents them from forming crystals. Zinc-resistant mutants display antigenic alterations in coat proteins. These results suggest that zinc complexes with rhinovirus coat proteins and alters them so that they cannot function as substrates for proteases or as reactants in the assembly of the virus particles.

Capsid↗
PubMed1983

Coat protein binding sites on RNA 1 of alfalfa mosaic virus.

The largest genome segment, RNA 1, of alfalfa mosaic virus forms complexes with viral coat protein. These complexes were subjected to digestion with ribonucleases T1 or A and filtered onto Millipore filters. Specific fragments were collected from the filters by phenol extraction. After electrophoretic separation in denaturing polyacrylamide gels, these fragments were sequenced. Besides extracistronic fragments originating from the 3'-terminal region of RNA 1, fragments were found originating from an intracistronic region of the RNA. A striking phenomenon is that the intracistronic fragments were not found when ribonuclease A was used to degrade RNA/protein complexes. The findings are in agreement with the postulation of Houwing and Jaspars (1978), that a conformational change at the 3' ends of the genome RNAs induced by the coat protein is a prerequisite to start an infection cycle.

Base Sequence↗
PubMed2002

Dissection of COPI and Arf1 dynamics in vivo and role in Golgi membrane transport.

Cytosolic coat proteins that bind reversibly to membranes have a central function in membrane transport within the secretory pathway. One well-studied example is COPI or coatomer, a heptameric protein complex that is recruited to membranes by the GTP-binding protein Arf1. Assembly into an electron-dense coat then helps in budding off membrane to be transported between the endoplasmic reticulum (ER) and Golgi apparatus. Here we propose and corroborate a simple model for coatomer and Arf1 activity based on results analysing the distribution and lifetime of fluorescently labelled coatomer and Arf1 on Golgi membranes of living cells. We find that activated Arf1 brings coatomer to membranes. However, once associated with membranes, Arf1 and coatomer have different residence times: coatomer remains on membranes after Arf1-GTP has been hydrolysed and dissociated. Rapid membrane binding and dissociation of coatomer and Arf1 occur stochastically, even without vesicle budding. We propose that this continuous activity of coatomer and Arf1 generates kinetically stable membrane domains that are connected to the formation of COPI-containing transport intermediates. This role for Arf1/coatomer might provide a model for investigating the behaviour of other coat protein systems within cells.

ADP-Ribosylation Factor 1↗
PubMed2005

Leucine-specific, functional interactions between human immunodeficiency virus type 1 Nef and adaptor protein complexes.

The human immunodeficiency virus type 1 virulence protein Nef interacts with the endosomal sorting machinery via a leucine-based motif. Similar sequences within the cytoplasmic domains of cellular transmembrane proteins bind to the adaptor protein (AP) complexes of coated vesicles to modulate protein traffic, but the molecular basis of the interactions between these motifs and the heterotetrameric complexes is controversial. To identify the target of the Nef leucine motif, the native sequence was replaced with either leucine- or tyrosine-based AP-binding sequences from cellular proteins, and the interactions with AP subunits were correlated with function. Tyrosine motifs predictably modulated the interactions between Nef and the mu subunits of AP-1, AP-2, and AP-3; heterologous leucine motifs caused little change in these interactions. Conversely, leucine motifs mediated a ternary interaction between Nef and hemicomplexes containing the sigma1 plus gamma subunits of AP-1 or the sigma3 plus delta subunits of AP-3, whereas tyrosine motifs did not. Similarly, only leucine motifs supported the Nef-mediated association of AP-1 and AP-3 with endosomal membranes in cells treated with brefeldin A. Functionally, Nef proteins containing leucine motifs down-regulated CD4 from the cell surface and enhanced viral replication, whereas those containing tyrosine motifs were inactive. Apparently, the interaction of Nef with the mu subunits of AP complexes is insufficient for function. A leucine-specific mode of interaction that likely involves AP hemicomplexes is further required for Nef activity. The mu and hemicomplex interactions may cooperate to yield high avidity binding of AP complexes to Nef. This binding likely underlies the unusual ability of Nef to induce the stabilization of these complexes on endosomal membranes, an activity that correlates with enhancement of viral replication.

Adaptor Protein Complex mu Subunits↗
PubMed2003

Dsl1p, an essential component of the Golgi-endoplasmic reticulum retrieval system in yeast, uses the same sequence motif to interact with different subunits of the COPI vesicle coat.

Dsl1p is required for Golgi-endoplasmic reticulum (ER) retrograde transport in yeast. It interacts with the ER resident protein Tip20p and with delta-COP, a subunit of coatomer, the coat complex of COPI vesicles. To test the significance of these interactions, we mapped the different binding sites and created mutant versions of Dsl1p and delta-COP, which are unable to bind directly to each other. Three domains were identified in Dsl1p: a Tip20p binding region within the N-terminal 200 residues, a highly acidic region in the center of Dsl1p containing crucial tryptophan residues that is required for binding to delta-COP and essential for viability, and an evolutionarily well conserved domain at the C terminus. Most importantly, Dsl1p uses the same central acidic domain to interact not only with delta-COP but also with alpha-COP. Strong interaction with alpha-COP requires the presence of comparable amounts of epsilon-COP or beta' -COP. Thus, the binding characteristics of Dsl1p resemble those of many accessory factors of the clathrin coat. They interact with different layers of the vesicle coat by using tandemly arranged sequence motifs, some of which have dual specificity.

Amino Acid Motifs↗
PubMed2006

Involvement of specific COPI subunits in protein sorting from the late endosome to the vacuole in yeast.

Although COPI function on the early secretory pathway in eukaryotes is well established, earlier studies also proposed a nonconventional role for this coat complex in endocytosis in mammalian cells. Here we present results that suggest an involvement for specific COPI subunits in the late steps of endosomal protein sorting in Saccharomyces cerevisiae. First, we found that carboxypeptidase Y (CPY) was partially missorted to the cell surface in certain mutants of the COPIB subcomplex (COPIb; Sec27, Sec28, and possibly Sec33), which indicates an impairment in endosomal transport. Second, integral membrane proteins destined for the vacuolar lumen (i.e., carboxypeptidase S [CPS1]; Fur4, Ste2, and Ste3) accumulated at an aberrant late endosomal compartment in these mutants. The observed phenotypes for COPIb mutants resemble those of class E vacuolar protein sorting (vps) mutants that are impaired in multivesicular body (MVB) protein sorting and biogenesis. Third, we observed physical interactions and colocalization between COPIb subunits and an MVB-associated protein, Vps27. Together, our findings suggest that certain COPI subunits could have a direct role in vacuolar protein sorting to the MVB compartment.

Cathepsin A↗
PubMed1985

Autoregulation of the bacteriophage P22 scaffolding protein gene.

During the formation of each bacteriophage P22 head, about 250 molecules of the product of gene 8, scaffolding protein, coassemble with and dictate correct assembly of the coat protein into a proper shell structure. At approximately the time that DNA is inserted inside the coat protein shell, all of the scaffolding protein molecules leave the structure. They remain active and participate in several subsequent rounds of shell assembly. Previous work has shown that scaffolding protein gene expression is affected by the head assembly process and has generated the hypothesis that unassembled scaffolding protein negatively modulates the expression of its own gene but that it lacks this activity when complexed with coat protein in proheads. To test this model, a P22 restriction fragment containing the scaffolding and coat protein genes was cloned under control of the lac promoter. These cloned genes were then expressed in an in vitro DNA-dependent transcription-translation reaction. The addition of purified scaffolding protein to this reaction resulted in reduced scaffolding protein synthesis relative to coat and tail protein synthesis to an extent and at a protein concentration that was consistent with the observed reduction in vivo. We conclude that scaffolding protein synthesis is autoregulated and that scaffolding protein is the only phage-coded protein required for this process. In addition, these experiments provide additional evidence that this autoregulation is posttranscriptional.

Base Sequence↗
PubMed2001

Rab1 interaction with a GM130 effector complex regulates COPII vesicle cis--Golgi tethering.

Members of the Rab family of small molecular weight GTPases regulate the fusion of transport intermediates to target membranes along the biosynthetic and endocytic pathways. We recently demonstrated that Rab1 recruitment of the tethering factor p115 into a cis-SNARE complex programs coat protein II vesicles budding from the endoplasmic reticulum (donor compartment) for fusion with the Golgi apparatus (acceptor compartment) (Allan BB, Moyer BD, Balch WE. Science 2000; 289: 444-448). However, the molecular mechanism(s) of Rab regulation of Golgi acceptor compartment function in endoplasmic reticulum to Golgi transport are unknown. Here, we demonstrate that the cis-Golgi tethering protein GM130, complexed with GRASP65 and other proteins, forms a novel Rab1 effector complex that interacts with activated Rab1-GTP in a p115-independent manner and is required for coat protein II vesicle targeting/fusion with the cis-Golgi. We propose a 'homing hypothesis' in which the same Rab interacts with distinct tethering factors at donor and acceptor membranes to program heterotypic membrane fusion events between transport intermediates and their target compartments.

Animals↗
PubMed2006

Key components of the fission machinery are interchangeable.

Brefeldin-A ADP-ribosylated substrate (BARS) and dynamin function in membrane fission in distinct intracellular transport pathways, but whether their functions are mechanistically similar is unclear. Here, we show that ARFGAP1, a GTPase-activating protein (GAP) for ADP-ribosylation factor 1 (ARF1), couples to either BARS or endophilin B for vesicle formation by the coat protein I (COPI) complex - a finding that reveals an unanticipated mechanistic flexibility in mammalian COPI transport. Because dynamin is coupled to endophilin A in vesicle formation by the clathrin-coat complex, our finding also predicts that dynamin and ARF GAPs are likely to be functional counterparts in membrane fission among different transport pathways that connect intracellular membrane compartments.

Acyltransferases↗
PubMed2000

Three ways to make a vesicle.

Cargo molecules have to be included in carrier vesicles of different forms and sizes to be transported between organelles. During this process, a limited set of proteins, including the coat proteins COPI, COPII and clathrin, carries out a programmed set of sequential interactions that lead to the budding of vesicles. A general model to explain the formation of coated vesicles is starting to emerge but the picture is more complex than we had imagined.

Animals↗
PubMed1986

Structure and assembly of turnip crinkle virus. II. Mechanism of reassembly in vitro.

Dissociation of turnip crinkle virus (TCV) at elevated pH and ionic strength produces free dimers of the coat protein and a ribonucleoprotein complex that contains the viral RNA, six coat-protein subunits, and the minor protein species, p80 (a covalently linked coat-protein dimer). This "rp-complex" is stable for several days in high salt at pH 8.5. Reassembly of TCV can be accomplished under physiological conditions, using isolated coat protein and either rp-complex or protein-free RNA. If rp-complex is used in reassembly, the same subunits remain bound to RNA on subsequent dissociation; if free RNA is used, rp-complex is regenerated. In both cases, the assembly is selective for viral RNA in competition experiments with heterologous RNA. Electron microscopy shows that assembly proceeds by continuous growth of a shell from an initiating structure, rather than by formation of distinct intermediates. We suggest that rp-complex is the initiating structure, suggest a model based on the organization of the TCV particle, and propose a mechanism for TCV assembly.

Capsid↗
PubMed2001

Similar subunit interactions contribute to assembly of clathrin adaptor complexes and COPI complex: analysis using yeast three-hybrid system.

Clathrin adaptor protein (AP) complexes are heterotetramers composed of two large, one medium, and one small subunits. By exploiting the yeast three-hybrid system, we have found that an interaction between the two large subunits of the AP-1 complex, gamma-adaptin and beta1-adaptin, is markedly enhanced in the presence of the small subunit, sigma1. Similarly, two large subunits of the AP-4 complex, epsilon-adaptin and beta4-adaptin, are found to interact with each other only in the presence of the small subunit, sigma4. Furthermore, we have found that an interaction between two large subunits of the COPI F subcomplex, gamma-COP and beta-COP, is detectable only in the presence of zeta-COP. Because these COPI subunits have common ancestral origins to the corresponding AP subunits, these three-hybrid data, taken together with the previous two-hybrid data, suggest that the AP complexes and the COPI F subcomplex assemble by virtue of similar subunit interactions.

Adaptor Protein Complex 1↗
PubMed2003

Depletion of the photosystem II core complex in mature tobacco leaves infected by the flavum strain of tobacco mosaic virus.

The flavum strain of Tobacco mosaic virus (TMV) differs from the wild-type (wt) virus by causing strong yellow and green mosaic in the systemically infected developing leaves, yellowing in the fully expanded leaves, and distinct malformations of chloroplasts in both types of infected tissues. Analysis of the thylakoid proteins of flavum strain-infected tobacco leaves indicated that the chlorosis in mature leaves was accompanied by depletion of the entire photosystem II (PSII) core complexes and the 33-kDa protein of the oxygen evolving complex. The only change observed in the thylakoid proteins of the corresponding wt TMV-infected leaves was a slight reduction of the alpha and beta subunits of the ATP synthase complex. The coat proteins of different yellowing strains of TMV are known to effectively accumulate inside chloroplasts, but in this work, the viral movement protein also was detected in association with the thylakoid membranes of flavum strain-infected leaves. The mRNAs of different enzymes involved in the chlorophyll biosynthesis pathway were not reduced in the mature chlorotic leaves. These results suggest that the chlorosis was not caused by reduction of pigment biosynthesis, but rather, by reduction of specific proteins of the PSII core complexes and by consequent break-down of the pigments.

Base Sequence↗
PubMed1975

Photo-incuced cross-linkage of gene-5 protein and bacteriophage fd DNA+.

The gene 5 protein, coded for by the bacteriophage fd, forms a complex with single stranded fd-DNA such that one gene 5 protein monomer interacts with four bases. Exposure of this complex to ultraviolet light results in the formation of covalent bonds between 25-30% of the gene 5 protein monomers which are bound to the DNA. In contrast, when the intact fd virion, which is a complex of coat protein and DNA, was exposed to ultraviolet irradiation, no detectable protein DNA cross-links were found.

Bacterial Proteins↗
PubMed2000

Oligomerization of peptides analogous to the cytoplasmic domains of coatomer receptors revealed by mass spectrometry.

Members of the p24 family of type I transmembrane proteins are involved in budding of coat protein type I (COPI)-coated vesicles. They serve as coat protein receptors, binding via their cytoplasmic domains to coatomer, a stable cytosolic protein complex that represents the major coat component of these vesicles. Experimental evidence suggest that p23, a member of the p24 family, binds to coatomer in an oligomeric state and that this binding triggers polymerization of the coat protein. Toward an understanding of this process at the molecular level, formation of noncovalent complexes and their relative stabilities were analyzed by Fourier transform ion cyclotron resonance mass spectrometry using nanoelectrospray ionization. Specificity and stability of oligomers formed were established to depend on characteristic peptide sequence motifs and were confirmed by mass spectrometric competition experiments with control peptides. Mutations in the peptide sequence caused decreased interaction and destabilization of the noncovalent complexes. The formation and relative stabilities of dimeric and tetrameric complexes were assessed to be formed by cytoplasmic tails of coatomer receptors. The direct molecular identification provided by mass spectrometry correlates well with biochemical results. Thus, electrospray ionization mass spectrometry proves to be a powerful tool to investigate physiologically relevant peptide complexes.

Amino Acid Sequence↗
PubMed2002

Differential requirements for COPI coats in formation of replication complexes among three genera of Picornaviridae.

Picornavirus RNA replication requires the formation of replication complexes (RCs) consisting of virus-induced vesicles associated with viral nonstructural proteins and RNA. Brefeldin A (BFA) has been shown to strongly inhibit RNA replication of poliovirus but not of encephalomyocarditis virus (EMCV). Here, we demonstrate that the replication of parechovirus 1 (ParV1) is partly resistant to BFA, whereas echovirus 11 (EV11) replication is strongly inhibited. Since BFA inhibits COPI-dependent steps in endoplasmic reticulum (ER)-Golgi transport, we tested a hypothesis that different picornaviruses may have differential requirements for COPI in the formation of their RCs. Using immunofluorescence and cryo-immunoelectron microscopy we examined the association of a COPI component, beta-COP, with the RCs of EMCV, ParV1, and EV11. EMCV RCs did not contain beta-COP. In contrast, beta-COP appeared to be specifically distributed to the RCs of EV11. In ParV1-infected cells beta-COP was largely dispersed throughout the cytoplasm, with some being present in the RCs. These results suggest that there are differences in the involvement of COPI in the formation of the RCs of various picornaviruses, corresponding to their differential sensitivity to BFA. EMCV RCs are likely to be formed immediately after vesicle budding from the ER, prior to COPI association with membranes. ParV1 RCs are formed from COPI-containing membranes but COPI is unlikely to be directly involved in their formation, whereas formation of EV11 RCs appears to be dependent on COPI association with membranes.

Animals↗