Fractionation of membrane vesicles from coliphage M13-infected Escherichia coli.

Membrane vesicles were prepared by osmotic lysis of spheroplasts from M13-infected Escherichia coli. Reduced nicotinamide adenine dinucleotide (NADH) oxidase (reduced NAD: oxidoreductase, EC 1.6.99.3) and Mg2+-Ca2+-activated adenosine triphosphatase (ATP phosphohydrolase, EC 3.6.1.3), which are normally localized to the inner surface of the cytoplasmic membrane, were 50% acceesible to their polar substrates in these vesicles. The major coat protein of coliphage M13 is also bound to the cytoplasmic membrane (prior to phage assembly) but with its antigenic sites exposed to the exterior of the cell. Antibody to M13 coat protein was used to fractionate membrane vesicles. Neither agglutinated nor unagglutinated vesicles had altered NADH oxidase and adenosine triphosphatase specific activities. This is inconsistent with such vesicles being a mixture of correctly oriented and completely inverted membrane sacs and suggests that NADH oxidase, adenosine triphosphatase, M13 coat protein, or all three proteins rearrange during vesicle preparation.     

The influenza virus M2 protein cytoplasmic tail interacts with the M1 protein and influences virus assembly at the site of virus budding

The cytoplasmic tail of the influenza A virus M2 proton-selective ion channel has been shown to be important for virus replication. Previous analysis of M2 cytoplasmic tail truncation mutants demonstrated a defect in incorporation of viral RNA (vRNA) into virions, suggesting a role for M2 in the recruitment of M1-vRNA complexes. To further characterize the effect of the M2 cytoplasmic tail mutations on virus assembly and budding, we constructed a series of alanine substitution mutants of M2 with mutations in the cytoplasmic tail, from residues 71 to 97. Mutant proteins M2-Mut1 and M2-Mut2, with mutations of residues 71 to 73 and 74 to 76, respectively, appeared to have the greatest effect on virus-like particle and virus budding, showing a defect in M1 incorporation. Mutant viruses containing M2-Mut1 and M2-Mut2 failed to replicate in multistep growth analyses on wild-type (wt) MDCK cells and were able to form plaques only on MDCK cells stably expressing wt M2 protein. Compared to wt M2 protein, M2-Mut1 and M2-Mut2 were unable to efficiently coimmunoprecipitate with M1. Furthermore, statistical analysis of planar sheets of membrane from cells infected by virus containing M2-Mut1 revealed a reduction in M1-hemagglutinin (HA) and M2-HA clustering as well as a severe loss of clustering between M1 and M2. These results suggest an essential, direct interaction between the cytoplasmic tail of M2 and M1 that promotes the recruitment of the internal viral proteins and vRNA to the plasma membrane for efficient virus assembly to occur.     

Nucleus-targeting domain of the matrix protein (M1) of influenza virus.

The matrix protein M1 of influenza virus A/WSN/33 was shown by immunofluorescent staining to be transported into the nuclei of transfected cells without requiring other viral proteins. We postulated the existence of a potential signal sequence at amino acids 101 to 105 (RKLKR) that is required for nuclear localization of the M1 protein. When CV1 cells were transfected with recombinant vectors expressing the entire M1 protein (amino acids 1 to 252) or just the first 112 N-terminal amino acids, both the complete M1 protein and the truncated M1 protein were transported to the nucleus. In contrast, expression in CV1 cells of vectors coding for M1 proteins with deletions from amino acids 77 to 202 or amino acids 1 to 134 resulted only in cytoplasmic immunofluorescent staining of these truncated M1 proteins without protein being transported to the nucleus. Moreover, no nuclear membrane translocation occurred when CV1 cells were transfected with recombinant vectors expressing M1 proteins with deletions of amino acids 101 to 105 or with substitution at amino acids 101 to 105 of SNLNS for RKLKR. Furthermore, a synthetic oligopeptide corresponding to M1 protein amino acids 90 to 108 was also transported into isolated nuclei derived from CV1 cells, whereas oligopeptides corresponding to amino acid sequences 25 to 40, 67 to 81, and 135 to 164 were not transported into the isolated cell nuclei. These data suggest that the amino acid sequence 101RKLKR105 is the nuclear localization signal of the M1 protein.     

Expression and membrane integration of SARS-CoV M protein

SARS-CoV M gene fragment was cloned and expressed as a recombinant protein fused with a V5 tag at the C-terminus in Vero E6 cells. In addition to un-glycosylated and glycosylated proteins, one product with smaller size initiated in-frame from the third Met residues probably through ribosomal re-initiation was also detected. Translation initiated in-frame from the third Met is unusual since the sequence around the first Met of SARS-CoV M protein contains the optimal consensus Kozak sequence. The function of this smaller translated product awaits further investigation. Similar to other N-glycosylated proteins, glycosylation of SARS-CoV M protein was occurred co-translationally in the presence of microsomes. The SARS-CoV M protein is predicted as a triple-spanning membrane protein lack of a conventional signal peptide. The second and third trans-membrane regions (a.a. 46–68 and 78–100) are predicted to be the primary type helices, which will be able to penetrate into membrane by themselves, while the first trans-membrane region (a.a. 14–36) is predicted to be the secondary type helix, which is considered to be stabilized by the interaction with other trans-membrane segments. As expected, the second and third trans-membrane regions were able to insert a cytoplasmic protein into the endoplasmic reticulum membrane more efficiently than the first one. These results should be important for the study of SARS-CoV morphogenesis. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Coordinated alterations in ribosomes and cytoplasmic membrane in sucrose-dependent, spectinomycin-resistant mutants of Escherichia coli.

Alterations in cytoplasmic membrane and ribosomes from sucrose-dependent spectinomycin-resistant (Sucd-Spcr) mutants of Escherichia coli, mutants that are resistant to spectinomycin in the presence of 20% sucrose but sensitive in the absence of sucrose, were studied. The protein composition of cytoplasmic membrane was analyzed by gel electrophoresis on polyacrylamide gel containing 8 M urea and 0.5% sodium dodecyl sulfate, which assured the reproducible separation of 28 protein bands. A major protein band, I-19, was missing in all cytoplasmic membrane preparations from 10 Sucd-Spcr mutants. Besides protein I-19, proteins I-13 and I-24 were missing in some mutants. On the other hand, the protein composition of cytoplasmic membrane from a sucrose-independent spectinomycin-resistant mutant was indistinguishable from that from the wild-type strain. The polypeptide synthetic activity of ribosomes from Sucd-Spcr mutants was resistant to spectinomycin. Studies on a revertant obtained from one of these mutants without any selection for sensitivity to spectinomycin revealed that a single mutation was responsible for both the ribosomal alteration, i.e., spectinomycin resistance, and the lack of protein I-19 in the cytoplasmic membrane. Studies on a transductant obtained with a Sucd-SPcr mutant as the donor also confirmed the single-mutation concept. It was concluded that in Sucd-SPcr mutants an alteration in the ribosomes caused the deletion of protein I-19 from cytoplasmic membrane.     

Interactions between M protein and other structural proteins of severe,acute respiratory syndrome-associated coronavirus

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) structural proteins (S, E, M, and NC) localize in different subcellular positions when expressed individually. However, SARS-CoV M protein is co-localized almost entirely with S, E, or NC protein when co-expressed in the cells. On the other hand, only partial co-localization was observed when S and E, S and NC, or E and NC were co-expressed in the cells. Interactions between SARS-CoV M and other structural proteins but not interactions between S and E, S and NC, or E and NC were further demonstrated by co-immunoprecipitation assay. These results indicate that SARS-CoV M protein, similar to the M proteins of other coronaviruses, plays a pivotal role in virus assembly. The cytoplasmic C-terminus domain of SARS-CoV M protein was responsible for binding to NC protein. Multiple regions of M protein interacted with E and S proteins. A model for the interactions between SARS-CoV M protein and other structural proteins is proposed. This study helps us better understand protein-protein interactions during viral assembly of SARS-CoV. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cytotoxic action of the individual membrane components of Mycoplasma arthritidis and M. fermentans on rat lymphocytes

Study of the toxic properties of the preparations obtained from M. arthritidis has revealed that the cytotoxic activity of M. arthritidis is mainly linked with the cytoplasmic membrane and, partially, with the cytoplasmic fraction. The membrane substances of M. fermentans and the products of its vital activity are toxic with respect to target cells, the component translocated into the culture medium consisting of globular proteins. Interaction of the cytoplasmic membranes of these Mycoplasma species, as well as of the fractions of M. fermentans globular proteins, with rat lymphocytes is accompanied by a cytodestructive effect and an increased permeability for toxic dyes.     

Chemical ligation of the influenza M2 protein for solid‐state NMR characterization of the cytoplasmic domain

Solid‐state NMR‐based structure determination of membrane proteins and large protein complexes faces the challenge of limited spectral resolution when the proteins are uniformly ¹³C‐labeled. A strategy to meet this challenge is chemical ligation combined with site‐specific or segmental labeling. While chemical ligation has been adopted in NMR studies of water‐soluble proteins, it has not been demonstrated for membrane proteins. Here we show chemical ligation of the influenza M2 protein, which contains a transmembrane (TM) domain and two extra‐membrane domains. The cytoplasmic domain, which contains an amphipathic helix (AH) and a cytoplasmic tail, is important for regulating virus assembly, virus budding, and the proton channel activity. A recent study of uniformly ¹³C‐labeled full‐length M2 by spectral simulation suggested that the cytoplasmic tail is unstructured. To further test this hypothesis, we conducted native chemical ligation of the TM segment and part of the cytoplasmic domain. Solid‐phase peptide synthesis of the two segments allowed several residues to be labeled in each segment. The post‐AH cytoplasmic residues exhibit random‐coil chemical shifts, low bond order parameters, and a surface‐bound location, thus indicating that this domain is a dynamic random coil on the membrane surface. Interestingly, the protein spectra are similar between a model membrane and a virus‐mimetic membrane, indicating that the structure and dynamics of the post‐AH segment is insensitive to the lipid composition. This chemical ligation approach is generally applicable to medium‐sized membrane proteins to provide site‐specific structural constraints, which complement the information obtained from uniformly ¹³C, ¹⁵N‐labeled proteins.     

Assembly of viral membranes: nature of the association of vesicular stomatitis virus proteins to membranes.

To explore the interaction of vesicular stomatitis virus (VSV) proteins with cellular membranes, we have isolated membranes from infected cells that have been radioactively pulse-labeled. We have found conditions of isolation that result in membrane preparation which contain primarily the VSV membrane protein (M) and glycoprotein (G). Both of these proteins are very firmly attached to membranes: conditions known to release peripherally associated membrane proteins from membranes (S. Razin, Biochim, Biophys. Acta 265:241-246, 1972; S. J. Singer, Annu. Rev. Biochem. 43:805-826, 1974; S. J. Singer and G. L. Nicholson, Science 175:720-731, 1972) are ineffective in detaching either the G or the M protein. The results of trypsin digestion of these membrane fractions suggest that the M protein resides primarily on one side, the cytoplasmic side of cellular membranes, whereas the glycoprotein has been transported to the lumen of the membrane vesicle. However, we present evidence that the glycoprotein is transmembranal and that approximately 3,000 daltons of one end of the molecule is on the cytoplasmic side of the membrane. We have also found that undenatured VSV M protein contains a trypsin-resistant core with a molecular weight of 22,000. This region of the M protein is trypsin-resistant regardless of its association with membranes.     

Complex formation among murine cytomegalovirus US22 proteins encoded by genes M139, M140, and M141

The murine cytomegalovirus (MCMV) proteins encoded by US22 genes M139, M140, and M141 function, at least in part, to regulate replication of this virus in macrophages. Mutant MCMV having one or more of these genes deleted replicates poorly in macrophages in culture and in the macrophage-dense environment of the spleen. In this report, we demonstrate the existence of stable complexes formed by the products of all three of these US22 genes, as well as a complex composed of the products of M140 and M141. These complexes form in the absence of other viral proteins; however, the pM140/pM141 complex serves as a requisite binding partner for the M139 gene products. Products from all three genes colocalize to a perinuclear region of the cell juxtaposed to or within the cis-Golgi region but excluded from the trans-Golgi region. Interestingly, expression of pM141 redirects pM140 from its predominantly nuclear residence to the perinuclear, cytoplasmic locale where these US22 proteins apparently exist in complex. Thus, complexing of these nonessential, early MCMV proteins likely confers a function(s) independent of each individual protein and important for optimal replication of MCMV in its natural host.     

Assembly of viral membranes. I. Association of vesicular stomatitis virus membrane proteins and membranes in a cell-free system.

We report here an in vitro system designed to study the interactions of vesicular stomatitis virus (VSV) proteins with cellular membranes. We have synthesized the VSV nucleocapsid (N) protein, nonstructural (NS) protein, glycoprotein (G protein), and membrane (M) protein in a wheat germ, cell-free, protein-synthesizing system directed by VSV 12 to 18S RNA. When incubated at low salt concentrations with purified cytoplasmic membranes derived from Chinese hamster ovary cells, the VSV M andG proteins bind to membranes, whereas the VSV N and NS proteins do not. The VSV M protein binds to membranes in low or high divalent cation concentrations, whereas binding of significant amounts of G protein requires at least 5 mM magnesium acetate concentrations.     

Transposition of domains between the M2 and HN viral membrane proteins results in polypeptides which can adopt more than one membrane orientation

The influenza A virus M2 polypeptide is a small integral membrane protein that does not contain a cleaved signal sequence, but is unusual in that it assumes the membrane orientation of a class I integral membrane protein with an NH2-terminal ectodomain and a COOH-terminal cytoplasmic tail. To determine the domains of M2 involved in specifying membrane orientation, hybrid genes were constructed and expressed in which regions of the M2 protein were linked to portions of the paramyxovirus HN and SH proteins, two class II integral membrane proteins that adopt the opposite orientation in membranes from M2. A hybrid protein (MgMH) consisting of the M2 NH2-terminal and membrane-spanning domains linked precisely to the HN COOH-terminal ectodomain was found in cells in two forms: integrated into membranes in the M2 topology or completely translocated across the endoplasmic reticulum membrane and ultimately secreted from the cell. The finding of a soluble form suggested that in this hybrid protein the anchor function of the M2 signal/anchor domain can be overridden. A second hybrid which contained the M2 NH2 terminus linked to the HN signal anchor and ectodomain (MgHH) was found in both the M2 and the HN orientation, suggesting that the M2 NH2 terminus was capable of reversing the topology of a class II membrane protein. The exchange of the M2 signal/anchor domain with that of SH resulted in a hybrid protein which assumed only the M2 topology. Thus, all these data suggest that the NH2-terminal 24 residues to M2 are important for directing the unusual membrane topology of the M2 protein. These data are discussed in relationship to the loop model for insertion of proteins into membranes and the role of charged residues as a factor in determining orientation.     

Evolutionary analysis of the influenza A virus M gene with comparison of the M1 and M2 proteins

Phylogenetic analysis of 42 membrane protein (M) genes of influenza A viruses from a variety of hosts and geographic locations showed that these genes have evolved into at least four major host-related lineages: (i) A/Equine/prague/56, which has the most divergent M gene; (ii) a lineage containing only H13 gull viruses; (iii) a lineage containing both human and classical swine viruses; and (iv) an avian lineage subdivided into North American avian viruses (including recent equine viruses) and Old World avian viruses (including avianlike swine strains). The M gene evolutionary tree differs from those published for other influenza virus genes (e.g., PB1, PB2, PA, and NP) but shows the most similarity to the NP gene phylogeny. Separate analyses of the M1 and M2 genes and their products revealed very different patterns of evolution. Compared with other influenza virus genes (e.g., PB2 and NP), the M1 and M2 genes are evolving relatively slowly, especially the M1 gene. The M1 and M2 gene products, which are encoded in different but partially overlapping reading frames, revealed that the M1 protein is evolving very slowly in all lineages, whereas the M2 protein shows significant evolution in human and swine lineages but virtually none in avian lineages. The evolutionary rates of the M1 proteins were much lower than those of M2 proteins and other internal proteins of influenza viruses (e.g., PB2 and NP), while M2 proteins showed less rapid evolution compared with other surface proteins (e.g., H3HA). Our results also indicate that for influenza A viruses, the evolution of one protein of a bicistronic gene can affect the evolution of the other protein.(ABSTRACT TRUNCATED AT 400 WORDS)     

The cytoplasmic tail of the influenza A virus M2 protein plays a role in viral assembly

The viral replication cycle concludes with the assembly of viral components to form progeny virions. For influenza A viruses, the matrix M1 protein and two membrane integral glycoproteins, hemagglutinin and neuraminidase, function cooperatively in this process. Here, we asked whether another membrane protein, the M2 protein, plays a role in virus assembly. The M2 protein, comprising 97 amino acids, possesses the longest cytoplasmic tail (54 residues) of the three transmembrane proteins of influenza A viruses. We therefore generated a series of deletion mutants of the M2 cytoplasmic tail by reverse genetics. We found that mutants in which more than 22 amino acids were deleted from the carboxyl terminus of the M2 tail were viable but grew less efficiently than did the wild-type virus. An analysis of the virions suggested that viruses with M2 tail deletions of more than 22 carboxy-terminal residues apparently contained less viral ribonucleoprotein complex than did the wild-type virus. These M2 tail mutants also differ from the wild-type virus in their morphology: while the wild-type virus is spherical, some of the mutants were filamentous. Alanine-scanning experiments further indicated that amino acids at positions 74 to 79 of the M2 tail play a role in virion morphogenesis and affect viral infectivity. We conclude that the M2 cytoplasmic domain of influenza A viruses plays an important role in viral assembly and morphogenesis.     

Cytoplasmic Compartmentalization of the Protein and Ribonucleic Acid Species of Vesicular Stomatitis Virus

The cytoplasmic sites of synthesis in L cells of the protein and ribonucleic acid species of vesicular stomatitis virus were studied by polyacrylamide gel electrophoresis after fractionation of membrane and other cytoplasmic components by the Caliguiri-Tamm technique. The viral spike protein (glycoprotein G) was found primarily associated with a smooth membrane fraction which is rich in plasma membrane; the G protein was also present in fractions containing rough endoplasmic reticulum. The nonglycosylated envelope protein S (also called M) was found in the smooth membrane fractions but was more abundant in endoplasmic reticulum-enriched fractions. Longer labeling resulted in detection of nucleoprotein N, as well as other minor nucleocapsid proteins L and NS₁, in the cellular membrane fractions. The N protein appeared to be made in membrane-free cytoplasm along with progeny ribonucleic acid and later became associated with membrane containing G and S viral proteins.     

Separation and characterization of the outer membrane of Pseudomonas aeruginosa.

A method is described for the preparation of outer and cytoplasmic membranes of Pseudomonas aeruginosa, and the outer membrane proteins characterized. Isolated outer and cytoplasmic membranes differed markedly in the content of 2-keto-3-deoxyoctonate (lipopolysaccharide) and phospholipid as well as in the localization of certain enzymes (NADH oxidase, succinate dehydrogenase, D-lactate dehydrogenase, malate dehydrogenase, and phospholipase), and also in the microscopic morphology. The outer membrane preparation showed activity neutralizing a certain bacteriocin or bacteriophages, whereas the cytoplasmic membrane preparation showed no neutralizing activity. The protein composition of membrane preparations from five different strains of P. aeruginosa [P14, M92 (PAO1), PAC1, P15, and M2008 (PAT)] were determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. More than 50 protein bands were detected in the cytoplasmic membrane preparation. The protein compositions of outer membranes from the five different strains were very similar: at least 6 major bands were found (apparent molecular weights: Band D, 50,000; band E, 45,000; band F, 33,000; bands G and H, 21,000; and band I, 8,000). The protein composition of outer membranes was affected by some physiological growth conditions. Some features of major outer membrane proteins were also studied. Band F showed anomalous migration on SDS polyacrylamide gel electrophoresis depending on the solubilizing conditions or pretreatment with TCA. Band I seemed to be a protein analogous to the lipoprotein which had been found in the outer membrane of Escherichia coli.     

The biology of colicin M

Abstract This communication summarizes our present knowledge of colicin M, an unusual member of the colicin group. The gene encoding colicin M, cma , has been sequenced and the protein isolated and purified. With a deduced molecular size of 29 453 Da, colicin M is the smallest of the known colicins. The polypeptide can be divided into functional domains for cell surface receptor binding, uptake into the cell, and killing activity. To kill, the colicin must enter from outside the cell. Colicin M blocks the biosynthesis of both peptidoglycan and O-antigen by inhibiting regeneration of the bactoprenyl-P carrier lipid. Autolysis occurs as a secondary effect following inhibition of peptidoglycan synthesis. Colicin M is the only colicin known to have such a mechanism of action. Immunity to this colicin is mediated by the cmi gene product, a protein of 13 890 Da. This cytoplasmic membrane protein confers immunity by binding to and thus neutralizing the colicin. Cmi shares properties with both immunity proteins of the pore-forming and the cytoplasmically active colicins. Genes for the colicin and immunity protein are found next to each other, but in opposite orientation, on pColM plasmids. The mechanism of colicin M release is not known.     

The biology of colicin M

This communication summarizes our present knowledge of colicin M, an unusual member of the colicin group. The gene encoding colicin M, cma, has been sequenced and the protein isolated and purified. With a deduced molecular size of 29,453 Da, colicin M is the smallest of the known colicins. The polypeptide can be divided into functional domains for cell surface receptor binding, uptake into the cell, and killing activity. To kill, the colicin must enter from outside the cell. Colicin M blocks the biosynthesis of both peptidoglycan and O-antigen by inhibiting regeneration of the bactoprenyl-P carrier lipid. Autolysis occurs as a secondary effect following inhibition of peptidoglycan synthesis. Colicin M is the only colicin known to have such a mechanism of action. Immunity to this colicin is mediated by the cmi gene product, a protein of 13,890 Da. This cytoplasmic membrane protein confers immunity by binding to and thus neutralizing the colicin. Cmi shares properties with both immunity proteins of the pore-forming and the cytoplasmically active colicins. Genes for the colicin and immunity protein are found next to each other, but in opposite orientation, on pColM plasmids. The mechanism of colicin M release is not known.     

Design and evolution of artificial M13 coat proteins

Using simple design and selective pressure, we have evolved an artificial M13 bacteriophage coat protein. M13 coat proteins first reside in the bacterial inner membrane and subsequently surround the DNA core of the assembled virus. The artificial coat protein (ACP) was designed and evolved to mimic both functions of the natural M13 coat proteins, but with an inverted orientation. ACP is a non-functional coat protein because it is not required for the production of phage particles. Instead, it incorporates into a phage coat which still requires all the natural coat proteins for structural integrity. In contrast with other M13 coat proteins, which can display polypeptides as amino-terminal fusions, ACP permits the carboxy-terminal display of large polypeptides. The results suggest that viruses can co-opt host membrane proteins to acquire new coat proteins and thus new functions. In particular, M13 bacteriophage can be engineered for new functions, such as carboxy-terminal phage display.