Multiple Membrane-spanning Segments Research Articles

Adaptive Laboratory Evolution for Multistress Tolerance, including Fermentability at High Glucose Concentrations in Thermotolerant Candida tropicalis

Candida tropicalis, a xylose-fermenting yeast, has the potential for converting cellulosic biomass to ethanol. Thermotolerant C. tropicalis X-17, which was isolated in Laos, was subjected to repetitive long-term cultivation with a gradual increase in temperature (RLCGT) in the presence of a high concentration of glucose, which exposed cells to various stresses in addition to the high concentration of glucose and high temperatures. The resultant adapted strain demonstrated increased tolerance to ethanol, furfural and hydroxymethylfurfural at high temperatures and displayed improvement in fermentation ability at high glucose concentrations and xylose-fermenting ability. Transcriptome analysis revealed the up-regulation of a gene for a glucose transporter of the major facilitator superfamily and genes for stress response and cell wall proteins. Additionally, hydropathy analysis revealed that three genes for putative membrane proteins with multiple membrane-spanning segments were also up-regulated. From these findings, it can be inferred that the up-regulation of genes, including the gene for a glucose transporter, is responsible for the phenotype of the adaptive strain. This study revealed part of the mechanisms of fermentability at high glucose concentrations in C. tropicalis and the results of this study suggest that RLCGT is an effective procedure for improving multistress tolerance.

Membrane-associated Ubiquitin Ligase Complex Containing gp78 Mediates Sterol-accelerated Degradation of 3-Hydroxy-3-methylglutaryl-coenzyme A Reductase

The endoplasmic reticulum (ER)-associated degradation (ERAD) pathway in the yeast Saccharomyces cerevisiae is mediated by two membrane-bound ubiquitin ligases, Doa10 and Hrd1. These enzymes are found in distinct multiprotein complexes that allow them to recognize and target a variety of substrates for proteasomal degradation. Although multiprotein complexes containing mammalian ERAD ubiquitin ligases likely exist, they have yet to be identified and characterized in detail. Here, we identify two ER membrane proteins, SPFH2 and TMUB1, as associated proteins of mammalian gp78, a membrane-bound ubiquitin ligase that bears significant sequence homology with mammalian Hrd1 and mediates sterol-accelerated ERAD of the cholesterol biosynthetic enzyme HMG-CoA reductase. Co-immunoprecipitation studies indicate that TMUB1 bridges SPFH2 to gp78 in ER membranes. The functional significance of these interactions is revealed by the observation that RNA interference (RNAi)-mediated knockdown of SPFH2 and TMUB1 blunts both the sterol-induced ubiquitination and degradation of endogenous reductase in HEK-293 cells. These studies mark the initial steps in the characterization of the mammalian gp78 ubiquitin ligase complex, the further elucidation of which may yield important insights into mechanisms underlying gp78-mediated ERAD.

Comparative genomics and function analysis on BI1 family

Bax inhibitor-1 (BI1) family contains six known genes in human. Some members of BI1 family have been proved to play important roles in cell death. Here we discuss the similarities and differences among the members of BI1 family by comparative genomics and proteomics, and report here the discovery of a novel member, tmbim1b, in Bos taurus. BI1 family is evolutionarily conserved as integral membrane proteins containing multiple membrane-spanning segments and predominantly localized to intracellular membranes, similar to Bcl-2 family proteins. They share multiple motifs and transcriptional factors within the promoter and the coding regions. They may represent regulators of cell death pathways, which are concluded from structure conservation of BI1 family.

The K15 Protein of Kaposi's Sarcoma-Associated Herpesvirus Recruits the Endocytic Regulator Intersectin 2 through a Selective SH3 Domain Interaction

Kaposi's sarcoma-associated herpesvirus, also known as human herpesvirus 8, is closely associated with several cancers including Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. The rightmost end of the KSHV genome encodes a protein, K15, with multiple membrane-spanning segments and an intracellular carboxy-terminal tail that contains several conserved motifs with the potential to recruit interaction domains (i.e., SH2, SH3, TRAF) of host cell proteins. K15 has been implicated in downregulating B cell receptor (BCR) signaling through its conserved motifs and may thereby play a role in maintaining viral latency and/or preventing apoptosis of the infected B cells. However, K15's mode of action is largely unknown. We have used mass spectrometry, domain and peptide arrays, and surface plasmon resonance to identify binding partners for a conserved proline-rich sequence (PPLP) in the K15 cytoplasmic tail. We show that the PPLP motif selectively binds the SH3-C domain of an endocytic adaptor protein, Intersectin 2 (ITSN2). This interaction can be observed both in vitro and in cells, where K15 and ITSN2 colocalize to discrete compartments within the B cell. The ability of K15 to associate with ITSN2 suggests a new role for the K15 viral protein in intracellular protein trafficking.

The cGMP-gated Channel and Related Glutamic Acid-rich Proteins Interact with Peripherin-2 at the Rim Region of Rod Photoreceptor Disc Membranes

The rod cGMP-gated channel is localized in the plasma membrane of rod photoreceptor outer segments, where it plays a central role in phototransduction. It consists of alpha- and beta-subunits that assemble into a heterotetrameric protein. Each subunit contains structural features characteristic of nucleotide-gated channels, including a cGMP-binding domain, multiple membrane-spanning segments, and a pore region. In addition, the beta-subunit has a large glutamic acid- and proline-rich region called GARP that is also expressed as two soluble protein variants. Using monoclonal antibodies in conjunction with immunoprecipitation, cross-linking, and electrophoretic techniques, we show that the cGMP-gated channel associates with the Na/Ca-K exchanger in the rod outer segment plasma membrane. This complex and soluble GARP proteins also interact with peripherin-2 oligomers in the rim region of outer segment disc membranes. These results suggest that channel/peripherin protein interactions mediated by the GARP part of the channel beta-subunit play a role in connecting the rim region of discs to the plasma membrane and in anchoring the channel.exchanger complex in the rod outer segment plasma membrane.

Pairing of the Nucleotide Binding Domains of the Transporter Associated with Antigen Processing

The transporter associated with antigen processing (TAP) comprises two structurally related subunits, TAP1 and TAP2, that form stable complexes in endoplasmic reticulum (ER) membranes. TAP complexes function in the translocation of peptides from the cytosol into the ER lumen for presentation by major histocompatibility complex class I molecules. Each TAP subunit contains an N-terminal membrane-spanning region with multiple membrane-spanning segments, and a C-terminal, cytosolic nucleotide binding region. To study the nature of the interactions occurring on the cytosolic face of TAP1/TAP2 complexes, we investigated quaternary associations mediated by two C-terminal fragments of human TAP1 (T1c, residues 452-748 and T1ctr, residues 472-748) and two C-terminal fragments of human TAP2 (T2c, residues 399-686 and T2ctr, residues 433-686). Each of these constructs contains the core nucleotide binding region as well as a long or short N-terminal extension. We show stable complex formation between T1c and T2c but not between T1ctr and T2ctr. The mechanistic implications of these results are discussed. We also show that each of the constructs except T1ctr interacts with wild type TAP1 and TAP2, indicating possibilities for homodimerization of TAP1 and TAP2, or of oligomerization of TAP1/TAP2 heterodimers on membranes.

Human FRAG1 Encodes a Novel Membrane-Spanning Protein That Localizes to Chromosome 11p15.5, a Region of Frequent Loss of Heterozygosity in Cancer

We have previously identified a chromosomal rearrangement between fibroblast growth factor receptor 2 (FGFR2) and a novel gene, FRAG1, in a rodent model of osteosarcoma. To assess the potential role of FRAG1 in disease further, we have isolated cDNA and genomic clones of human FRAG1. Sequence analysis of the cDNA revealed the presence of an insertion not contained in the original FRAG1 sequence. This insertion in human FRAG1 encoded a region highly homologous to and immediately following the first 55 amino acids of the protein, indicating the presence of a repetitive domain within FRAG1, designated the FRAG1 homology (FH) domain. Analysis of FRAG1 gene structure revealed that the FH domains were encoded by tandem duplicated exons. Database searches identified several transmembrane proteins displaying homology to the FH domain of FRAG1. In addition, hydropathy analysis predicted FRAG1 to encode an integral membrane protein with multiple membrane-spanning segments. FRAG1 mRNA was ubiquitously expressed in human adult tissues and several tumor cell lines at varying levels of abundance. Human FRAG1 was mapped by fluorescence in situ hybridization and radiation hybrid analysis to chromosome 11 at band p15.5, a region implicated in Beckwith–Wiedemann syndrome and a region of frequent loss of heterozygosity in multiple tumor types. These results suggest that FRAG1 may be a useful candidate gene for genetic disorders associated with alterations at 11p15.5.

Bax Inhibitor-1, a Mammalian Apoptosis Suppressor Identified by Functional Screening in Yeast

The mammalian proapoptotic protein Bax confers a lethal phenotype when expressed in yeast. By exploiting this phenotype, we have identified a novel human Bax inhibitor, BI-1. BI-1 is an evolutionarily conserved integral membrane protein containing multiple membrane-spanning segments and is predominantly localized to intracellular membranes, similar to Bcl-2 family proteins. Moreover, BI-1 can interact with Bcl-2 and Bcl-X L but not Bax or Bak, as demonstrated by in vivo cross-linking and coimmunoprecipitation studies. When overexpressed in mammalian cells, BI-1 suppressed apoptosis induced by Bax, etoposide, staurosporine, and growth factor deprivation, but not by Fas (CD95). Conversely, BI-1 antisense induced apoptosis. BI-1 thus represents a new type of regulator of cell death pathways controlled by Bcl-2 and Bax.

Stimulation of NF-kappa B-mediated transcription by mutant derivatives of the latent membrane protein of Epstein-Barr virus

The latent membrane protein (LMP) of Epstein-Barr virus contributes to the immortalizing activity of the virus in primary, human B lymphocytes, but its mechanism of function is unknown. LMP is expressed at the plasma membrane and may act by influencing the signalling pathways of infected cells. LMP increases transcription of reporter plasmids that are responsive to members of the NF-kappa B/Rel family of transcription factors (M.-L. Hammarskjold and M. C. Simurda, J. Virol. 66:6496-6501, 1992, and A. Krikos, C. D. Laherty, and V. M. Dixit, J. Biol. Chem. 267:17971-17976, 1992). We measured the stimulation of the activity of a reporter plasmid by LMP in Jurkat and 293 cells in transfection experiments. Expression of LMP stimulated plasmids that contained kappa B enhancer elements but not plasmids that lacked the elements. In 293 cells, expression of the NF-kappa B inhibitor, I kappa B-alpha, reduced the stimulatory activity of LMP. We used deletional analysis to map the domains of LMP that are required for its activity in 293 cells. Wild-type LMP stimulated NF-kappa B by a factor of 20 to 30, while mutant derivatives of LMP that lack oncogenic activity stimulated NF-kappa B by a factor of 3. The multiple membrane-spanning segments together with the carboxy-terminal 55 amino acid residues of LMP were required for its maximal stimulatory function. Residues within its cytoplasmic amino terminus were not required for LMP's stimulation of NF-kappa B. We tested also for stimulation of NF-kappa B activity in cell lines known to support phenotypic changes mediated by expression of LMP. LMP stimulated little NF-kappa B activity in HEp2 cells and no detectable NF-kappa B activity in BALB/3T3 cells. The LMP stimulation of NF-kappa B factors that occurs in some cell lines provides a useful and biochemically tractable assay for determining the function of LMP.

Nucleotide sequence of the yeast STE14 gene, which encodes farnesylcysteine carboxyl methyltransferase, and demonstration of its essential role in a-factor export.

Eukaryotic proteins initially synthesized with a C-terminal CAAX motif (C is Cys, A is aliphatic, and X can be one of several amino acids) undergo a series of modifications involving isoprenylation of the Cys residue, proteolysis of AAX, and alpha-carboxyl methyl esterification of the newly formed isoprenyl cysteine. We have previously demonstrated that STE14 encodes the enzyme which mediates carboxyl methylation of the Saccharomyces cerevisiae CAAX proteins a-factor, RAS1, and RAS2. Here we report the nucleotide sequence of STE14, which indicates that STE14 encodes a protein of 239 amino acids, predicted to contain multiple membrane-spanning segments. Mapping data indicate that STE14 resides on chromosome IV, tightly linked to ADE8. By analysis of ste14 null alleles, we demonstrated that MATa ste14 mutants are unable to mate but are viable and exhibit no apparent growth defects. Additional analysis of ste14 ras 1 and ste14 ras2 double mutants, which grow normally, reinforces our previous conclusion that RAS function is not significantly influenced by its methylation status. We examine a-factor biogenesis in a ste14 null mutant by metabolic labeling and immunoprecipitation and demonstrate that although proteolytic processing and membrane localization of a-factor are normal, the ste14 null mutant exhibits a profound block in a-factor export. This observation suggests that the methyl group is likely to be a critical recognition determinant for the a-factor transporter, STE6, thus providing insight into the substrate specificity of STE6 and also supporting the hypothesis that carboxyl methylation can have a dramatic impact on protein-protein interactions.

Nucleotide Sequence of the Yeast STE14 Gene, Which Encodes Farnesylcysteine Carboxyl Methyltransferase, and Demonstration of its Essential Role in a-Factor Export

Eukaryotic proteins initially synthesized with a C-terminal CAAX motif (C is Cys, A is aliphatic, and X can be one of several amino acids) undergo a series of modifications involving isoprenylation of the Cys residue, proteolysis of AAX, and alpha-carboxyl methyl esterification of the newly formed isoprenyl cysteine. We have previously demonstrated that STE14 encodes the enzyme which mediates carboxyl methylation of the Saccharomyces cerevisiae CAAX proteins a-factor, RAS1, and RAS2. Here we report the nucleotide sequence of STE14, which indicates that STE14 encodes a protein of 239 amino acids, predicted to contain multiple membrane-spanning segments. Mapping data indicate that STE14 resides on chromosome IV, tightly linked to ADE8. By analysis of ste14 null alleles, we demonstrated that MATa ste14 mutants are unable to mate but are viable and exhibit no apparent growth defects. Additional analysis of ste14 ras 1 and ste14 ras2 double mutants, which grow normally, reinforces our previous conclusion that RAS function is not significantly influenced by its methylation status. We examine a-factor biogenesis in a ste14 null mutant by metabolic labeling and immunoprecipitation and demonstrate that although proteolytic processing and membrane localization of a-factor are normal, the ste14 null mutant exhibits a profound block in a-factor export. This observation suggests that the methyl group is likely to be a critical recognition determinant for the a-factor transporter, STE6, thus providing insight into the substrate specificity of STE6 and also supporting the hypothesis that carboxyl methylation can have a dramatic impact on protein-protein interactions.

The membrane-bound proteins of periplasmic permeases form a complex. Identification of the histidine permease HisQMP complex

The membrane-bound proteins of periplasmic transport systems have been hypothesized to form a complex with relatively little experimental support. Here we present experimental evidence that HisQ, HisM, and HisP, the membrane-bound proteins of the periplasmic histidine transport system of Salmonella typhimurium, form such a complex. We have developed antibodies specific to each of these proteins to aid in their characterization. Extractions with urea, alkaline pH, or Triton X-114 show that HisQ and HisM are integral membrane proteins. By these tests HisP displays an unusual behavior, being associated with the membrane whether or not HisQ and HisM are present and despite its hydrophilic sequence. However, the nature of HisPs interaction with the membrane is shown to vary depending on the presence of HisQ and HisM. In their absence, HisP is somewhat peripherally associated with the membrane, while in their presence it binds much more tightly, indicating that it forms a complex in association with HisQ and HisM. This is demonstrated by the coimmunoprecipitation of all three proteins by antibodies directed against any one of them. Chemical cross-linking allowed the characterization of the subunit stoichiometry of the complex as two HisPs to one HisQ and one HisM. Within this complex all three proteins probably contact each other and the two HisPs form a dimer. We hypothesize that HisQ and HisM with their multiple membrane-spanning segments form a "channel" within which the HisP subunits are located.