scholarly journals The Transmembrane Domain of the Severe Acute Respiratory Syndrome Coronavirus ORF7b Protein Is Necessary and Sufficient for Its Retention in the Golgi Complex

2008 ◽  
Vol 82 (19) ◽  
pp. 9477-9491 ◽  
Author(s):  
Scott R. Schaecher ◽  
Michael S. Diamond ◽  
Andrew Pekosz
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Golgi Complex ◽  
Transmembrane Domain ◽  
Chimeric Protein ◽  
Integral Membrane Protein ◽  
Reading Frame ◽  
Mutant Proteins ◽  
Golgi Compartment ◽  
Carboxy Terminal ◽  
Necessary And Sufficient

ABSTRACT The severe acute respiratory syndrome coronavirus (SARS-CoV) ORF7b (also called 7b) protein is an integral membrane protein that is translated from a bicistronic open reading frame encoded within subgenomic RNA 7. When expressed independently or during virus infection, ORF7b accumulates in the Golgi compartment, colocalizing with both cis- and trans-Golgi markers. To identify the domains of this protein that are responsible for Golgi localization, we have generated a set of mutant proteins and analyzed their subcellular localizations by indirect immunofluorescence confocal microscopy. The N- and C-terminal sequences are dispensable, but the ORF7b transmembrane domain (TMD) is essential for Golgi compartment localization. When the TMD of human CD4 was replaced with the ORF7b TMD, the resulting chimeric protein localized to the Golgi complex. Scanning alanine mutagenesis identified two regions in the carboxy-terminal portion of the TMD that eliminated the Golgi complex localization of the chimeric CD4 proteins or ORF7b protein. Collectively, these data demonstrate that the Golgi complex retention signal of the ORF7b protein resides solely within the TMD.

scholarly journals Vaccinia Virus F1L Protein Is a Tail-Anchored Protein That Functions at the Mitochondria To Inhibit Apoptosis

2005 ◽  
Vol 79 (2) ◽  
pp. 1084-1098 ◽  
Author(s):  
Tara L. Stewart ◽  
Shawn T. Wasilenko ◽  
Michele Barry
Keyword(s):  
Amino Acid ◽  
Vaccinia Virus ◽  
Transmembrane Domain ◽  
In Vitro Transcription ◽  
Translation System ◽  
Reading Frame ◽  
Protease Digestion ◽  
Necessary And Sufficient ◽  
Virus Survival

ABSTRACT Members of the poxvirus family encode multiple immune evasion proteins, including proteins that regulate apoptosis. We recently identified one such protein, F1L, encoded by vaccinia virus, the prototypic member of the poxvirus family. F1L localizes to the mitochondria and inhibits apoptosis by interfering with the release of cytochrome c, the pivotal commitment step in the apoptotic cascade. Sequence analysis of the F1L open reading frame revealed a C-terminal motif composed of a 12-amino-acid transmembrane domain flanked by positively charged lysines, followed by an 8-amino-acid hydrophilic tail. By generating a series of F1L deletion constructs, we show that the C-terminal domain is necessary and sufficient for localization of F1L to the mitochondria. In addition, mutation of lysines 219 and 222 downstream of the C-terminal transmembrane domain resulted in altered localization of F1L to the endoplasmic reticulum. Using F1L protein generated in an in vitro transcription-translation system, we found that F1L was posttranslationally inserted into mitochondria and tightly associated with mitochondrial membranes as demonstrated by resistance to alkaline extraction. Sensitivity to protease digestion showed that the N terminus of F1L was exposed to the cytoplasm. Utilizing various F1L deletion constructs, we found that F1L localization to the mitochondria was necessary to inhibit apoptosis, since constructs that no longer localized to the mitochondria had reduced antiapoptotic ability. Our studies show that F1L is a new member of the tail-anchored protein family that localizes to mitochondria during virus infection and inhibits apoptosis as a means to enhance virus survival.

scholarly journals Characterization of kinectin, a kinesin-binding protein: primary sequence and N-terminal topogenic signal analysis.

1995 ◽  
Vol 6 (2) ◽  
pp. 171-183 ◽  
Author(s):  
H Yu ◽  
C V Nicchitta ◽  
J Kumar ◽  
M Becker ◽  
I Toyoshima ◽  
...  
Keyword(s):  
Amino Acids ◽  
Transmembrane Domain ◽  
Binding Protein ◽  
Coiled Coil ◽  
Integral Membrane Protein ◽  
In Vitro Translation ◽  
Predicted Amino Acid Sequence ◽  
Hydrophobic Region ◽  
Reading Frame

Kinectin is a kinesin-binding protein (Toyoshima et al., 1992) that is required for kinesin-based motility (Kumar et al., 1995). A kinectin cDNA clone containing a 4.7-kilobase insert was isolated from an embryonic chick brain cDNA library by immunoscreening with a panel of monoclonal antibodies. The cDNA contained an open reading frame of 1364 amino acids encoding a protein of 156 kDa. A bacterially expressed product of the full length cDNA bound purified kinesin. Transient expression in CV-1 cells gave an endoplasmic reticulum distribution that depended upon the N-terminal domain. Analysis of the predicted amino acid sequence indicated a highly hydrophobic near N-terminal stretch of 28 amino acids and a large portion (326-1248) of predicted alpha helical coiled coils. The 30-kDa fragment containing the N-terminal hydrophobic region was produced by cell-free in vitro translation and found to assemble with canine pancreas rough microsomes. Cleavage of the N terminus was not observed confirming its role as a potential transmembrane domain. Thus, the kinectin cDNA encodes a cytoplasmic-oriented integral membrane protein that binds kinesin and is likely to be a coiled-coil dimer.

scholarly journals Yos1p Is a Novel Subunit of the Yip1p–Yif1p Complex and Is Required for Transport between the Endoplasmic Reticulum and the Golgi Complex

2005 ◽  
Vol 16 (4) ◽  
pp. 1673-1683 ◽  
Author(s):  
Matthew Heidtman ◽  
Catherine Z. Chen ◽  
Ruth N. Collins ◽  
Charles Barlowe
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Complex ◽  
Secretory Pathway ◽  
Integral Membrane Protein ◽  
Rab Gtpases ◽  
Reading Frame ◽  
Multicopy Suppressor ◽  
Transport Vesicles ◽  
Identification And Characterization

Yeast Yip1p is a member of a conserved family of transmembrane proteins that interact with Rab GTPases. Previous studies also have indicated a role for Yip1p in the biogenesis of endoplasmic reticulum (ER)-derived COPII transport vesicles. In this report, we describe the identification and characterization of the uncharacterized open reading frame YER074W-A as a novel multicopy suppressor of the thermosensitive yip1-4 strain. We have termed this gene Yip One Suppressor 1 (YOS1). Yos1p is essential for growth and for function of the secretory pathway; depletion or inactivation of Yos1p blocks transport between the ER and the Golgi complex. YOS1 encodes an integral membrane protein of 87 amino acids that is conserved in eukaryotes. Yos1p localizes to ER and Golgi membranes and is efficiently packaged into ER-derived COPII transport vesicles. Yos1p associates with Yip1p and Yif1p, indicating Yos1p is a novel subunit of the Yip1p–Yif1p complex.

scholarly journals The Cytoplasmic Tail of Infectious Bronchitis Virus E Protein Directs Golgi Targeting

2002 ◽  
Vol 76 (3) ◽  
pp. 1273-1284 ◽  
Author(s):  
Emily Corse ◽  
Carolyn E. Machamer
Keyword(s):  
Golgi Complex ◽  
Infectious Bronchitis Virus ◽  
Transmembrane Domain ◽  
Brefeldin A ◽  
Cytoplasmic Tail ◽  
Reporter Protein ◽  
Infectious Bronchitis ◽  
E Protein ◽  
Necessary And Sufficient ◽  
Golgi Matrix

ABSTRACT We have previously shown that the E protein of the coronavirus infectious bronchitis virus (IBV) is localized to the Golgi complex when expressed exogenously from cDNA. Here, we report that neither the transmembrane domain nor the short lumenal domain of IBV E is required for Golgi targeting. However, an N-terminal truncation containing only the cytoplasmic domain (CTE) was efficiently localized to the Golgi complex, and this domain could retain a reporter protein in the Golgi. Thus, the cytoplasmic tail of the E protein is necessary and sufficient for Golgi targeting. The IBV E protein is palmitoylated on one or two cysteine residues adjacent to its transmembrane domain, but palmitoylation was not required for proper Golgi targeting. Using C-terminal truncations, we determined that the IBV E Golgi targeting information is present between tail amino acids 13 and 63. Upon treatment with brefeldin A, both the E and CTE proteins redistributed to punctate structures that colocalized with the Golgi matrix proteins GM130 and p115 instead of being localized to the endoplasmic reticulum like Golgi glycosylation enzymes. This suggests that IBV E is associated with the Golgi matrix through interactions of its cytoplasmic tail and may have interesting implications for coronavirus assembly in early Golgi compartments.

scholarly journals GxxxG Motif of Severe Acute Respiratory Syndrome Coronavirus Spike Glycoprotein Transmembrane Domain Is Not Involved in Trimerization and Is Not Important for Entry

2007 ◽  
Vol 81 (15) ◽  
pp. 8352-8355 ◽  
Author(s):  
Jeroen Corver ◽  
Rene Broer ◽  
Puck van Kasteren ◽  
Willy Spaan
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Transmembrane Domain ◽  
Vital Role ◽  
Sars Coronavirus ◽  
Spike Glycoprotein ◽  
S Protein ◽  
Mutant Proteins ◽  
Gxxxg Motif

ABSTRACT Recently, a paper was published in which it was proposed that the GxxxG motif of the severe acute respiratory syndrome (SARS) coronavirus spike (S) protein transmembrane domain plays a vital role in oligomerization of the protein (E. Arbely, Z. Granot, I. Kass, J. Orly, and I. T. Arkin, Biochemistry 45:11349-11356, 2006). Here, we show that the GxxxG motif is not involved in SARS S oligomerization by trimerization analysis of S GxxxG mutant proteins. In addition, the capability of S to mediate entry of SARS S-pseudotyped particles overall was affected moderately in the mutant proteins, also arguing for a nonvital role for the GxxxG motif in SARS coronavirus entry.

scholarly journals A palindromic regulatory site within vertebrate GATA-1 promoters requires both zinc fingers of the GATA-1 DNA-binding domain for high-affinity interaction.

1996 ◽  
Vol 16 (5) ◽  
pp. 2238-2247 ◽  
Author(s):  
C D Trainor ◽  
J G Omichinski ◽  
T L Vandergon ◽  
A M Gronenborn ◽  
G M Clore ◽  
...  
Keyword(s):  
Specific Binding ◽  
Gene Promoters ◽  
Recognition Sequence ◽  
Erythroid Precursor ◽  
Mutant Proteins ◽  
Amino Terminal ◽  
Canonical Motif ◽  
Carboxy Terminal ◽  
Necessary And Sufficient ◽  
Affinity Interaction

GATA-1, a transcription factor essential for the development of the erythroid lineage, contains two adjacent highly conserved zinc finger motifs. The carboxy-terminal finger is necessary and sufficient for specific binding to the consensus GATA recognition sequence: mutant proteins containing only the amino-terminal finger do not bind. Here we identify a DNA sequence (GATApal) for which the GATA-1 amino-terminal finger makes a critical contribution to the strength of binding. The site occurs in the GATA-1 gene promoters of chickens, mice, and humans but occurs very infrequently in other vertebrate genes known to be regulated by GATA proteins. GATApal is a palindromic site composed of one complete [(A/T)GATA(A/G)] and one partial (GAT) canonical motif. Deletion of the partial motif changes the site to a normal GATA site and also reduces by as much as eightfold the activity of the GATA-1 promoter in an erythroid precursor cell. We propose that GATApal is important for positive regulation of GATA-1 expression in erythroid cells.

scholarly journals Erf2, a Novel Gene Product That Affects the Localization and Palmitoylation of Ras2 in Saccharomyces cerevisiae

1999 ◽  
Vol 19 (10) ◽  
pp. 6775-6787 ◽  
Author(s):  
Doug J. Bartels ◽  
David A. Mitchell ◽  
Xiangwen Dong ◽  
Robert J. Deschenes
Keyword(s):  
Subcellular Localization ◽  
Integral Membrane Protein ◽  
Subcellular Fractionation ◽  
Growth Defect ◽  
Loss Of Function ◽  
Reading Frame ◽  
Rich Domain ◽  
Carboxy Terminal ◽  
The Relationship ◽  
Yeast Genetic

ABSTRACT Plasma membrane localization of Ras requires posttranslational addition of farnesyl and palmitoyl lipid moieties to a C-terminal CaaX motif (C is cysteine, a is any aliphatic residue, X is the carboxy terminal residue). To better understand the relationship between posttranslational processing and the subcellular localization of Ras, a yeast genetic screen was undertaken based on the loss of function of a palmitoylation-dependentRAS2 allele. Mutations were identified in an uncharacterized open reading frame (YLR246w) that we have designated ERF2 and a previously described suppressor of hyperactive Ras, SHR5. ERF2 encodes a 41-kDa protein with four predicted transmembrane (TM) segments and a motif consisting of the amino acids Asp-His-His-Cys (DHHC) within a cysteine-rich domain (CRD), called DHHC-CRD. Mutations within the DHHC-CRD abolish Erf2 function. Subcellular fractionation and immunolocalization experiments reveal that Erf2 tagged with a triply iterated hemagglutinin epitope is an integral membrane protein that colocalizes with the yeast endoplasmic reticulum marker Kar2. Strains lacking ERF2 are viable, but they have a synthetic growth defect in the absence of RAS2 and partially suppress the heat shock sensitivity resulting from expression of the hyperactiveRAS2(V19) allele. Ras2 proteins expressed in anerf2Δ strain have a reduced level of palmitoylation and are partially mislocalized to the vacuole. Based on these observations, we propose that Erf2 is a component of a previously uncharacterized Ras subcellular localization pathway. Putative members of an Erf2 family of proteins have been uncovered in yeast, plant, worm, insect, and mammalian genome databases, suggesting that Erf2 plays a role in Ras localization in all eucaryotes.

scholarly journals An uncleaved glycosylphosphatidylinositol signal mediates Ca2+-sensitive protein degradation

1996 ◽  
Vol 317 (2) ◽  
pp. 533-540
Author(s):  
Peter C. PAULY ◽  
Claudette KLEIN
Keyword(s):  
Endoplasmic Reticulum ◽  
Transmembrane Domain ◽  
Signal Sequence ◽  
Interleukin 2 ◽  
Chimeric Protein ◽  
Integral Membrane Protein ◽  
Marker Enzymes ◽  
Gpi Anchor ◽  
Percoll Gradients ◽  
Anchor Sequence

Inv-gp80 is a chimeric protein which contains a signal for the attachment of a glycosylphosphatidylinositol (GPI) anchor. When expressed in Dictyostelium discoideum, this protein fails to become GPI anchored and is retained within the cell as an integral membrane protein. We have compared the subcellular localization and degradation of Inv-gp80 with that of its intracellular but soluble counterpart, Inv-gp80sc. Inv-gp80sc lacks the hydrophobic C-terminal 22 amino acids of Inv-gp80. The N-linked oligosaccharides of both Inv-gp80 and Inv-gp80sc remained sensitive to endoglycosidase H, and both proteins co-fractionated with endoplasmic reticulum marker enzymes on Percoll gradients. Under normal conditions, Inv-gp80 displayed a half-life (t½) of 90 min, while Inv-gp80sc displayed a t½ of 120 min. The degradation of both proteins required ATP, was inhibited by tosyl phenylalanylchloromethane (Tos-Phe-CH2Cl) and was insensitive to inhibitors of lysosomal function. While depletion of Ca2+ from the endoplasmic reticulum had no effect on the degradation of Inv-gp80sc, it stimulated the degradation of Inv-gp80. When the GPI anchor signal sequence of Inv-gp80 was replaced with the transmembrane domain of the interleukin-2 receptor, the degradation of the protein was no longer influenced by Ca2+ fluxes. The data suggest that while the GPI anchor sequence of Inv-gp80 does not contain determinants regulating the degradation of the protein under basal conditions, it targets Inv-gp80 for rapid degradation under conditions where Ca2+ is depleted from the endoplasmic reticulum.

scholarly journals Sorting of yeast alpha 1,3 mannosyltransferase is mediated by a lumenal domain interaction, and a transmembrane domain signal that can confer clathrin-dependent Golgi localization to a secreted protein.

1995 ◽  
Vol 6 (7) ◽  
pp. 809-824 ◽  
Author(s):  
T R Graham ◽  
V A Krasnov
Keyword(s):  
Golgi Complex ◽  
Fusion Proteins ◽  
Transmembrane Domain ◽  
Signal Sequence ◽  
Integral Membrane Protein ◽  
Retention Mechanism ◽  
Secreted Protein ◽  
Golgi Localization ◽  
In The Wild ◽  
Lumenal Domain

alpha 1,3 mannosyltransferase (Mnn1p) is a type II integral membrane protein that is localized to the yeast Golgi complex. We have examined the signals within Mnn1p that mediate Golgi localization by expression of fusion proteins comprised of Mnn1p and the secreted protein invertase. The N-terminal transmembrane domain (TMD) of Mnn1p is sufficient to localize invertase to the Golgi complex by a mechanism that is not saturable by approximately 15-20 fold overexpression. Furthermore, the TMD-mediated localization mechanism is clathrin dependent, as an invertase fusion protein bearing only the Mnn1p TMD is mislocalized to the plasma membrane of a clathrin heavy chain mutant. The Mnn1-invertase fusion proteins are not retained in the Golgi complex as efficiently as Mnn1p, suggesting that other signals may be present in the wild-type protein. Indeed, the Mnn1p lumenal domain (Mnn1-s) is also localized to the Golgi complex when expressed as a functional, soluble protein by exchanging its TMD for a cleavable signal sequence. In contrast to the Mnn1-invertase fusion proteins, overexpression of Mnn1-s saturates its retention mechanism, and results in the partial secretion of this protein. These data indicate that Mnn1p has separable Golgi localization signals within both its transmembrane and lumenal domains.

scholarly journals The ORF7b Protein of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) Is Expressed in Virus-Infected Cells and Incorporated into SARS-CoV Particles

2006 ◽  
Vol 81 (2) ◽  
pp. 718-731 ◽  
Author(s):  
Scott R. Schaecher ◽  
Jason M. Mackenzie ◽  
Andrew Pekosz
Keyword(s):  
Stop Codon ◽  
Integral Membrane Protein ◽  
Biochemical Properties ◽  
Accessory Protein ◽  
Coding Region ◽  
Reading Frame ◽  
Golgi Compartment ◽  
Infected Cells ◽  
Viral Accessory Protein

ABSTRACT Coronavirus replication is facilitated by a number of highly conserved viral proteins. The viruses also encode accessory genes, which are virus group specific and believed to play roles in virus replication and pathogenesis in vivo. Of the eight putative accessory proteins encoded by the severe acute respiratory distress syndrome associated coronavirus (SARS-CoV), only two—open reading frame 3a (ORF3a) and ORF7a—have been identified in virus-infected cells to date. The ORF7b protein is a putative viral accessory protein encoded on subgenomic (sg) RNA 7. The ORF7b initiation codon overlaps the ORF7a stop codon in a −1 shifted ORF. We demonstrate that the ORF7b protein is expressed in virus-infected cell lysates and from a cDNA encoding the gene 7 coding region, indicating that the sgRNA7 is bicistronic. The translation of ORF7b appears to be mediated by ribosome leaky scanning, and the protein has biochemical properties consistent with that of an integral membrane protein. ORF7b localizes to the Golgi compartment and is incorporated into SARS-CoV particles. We therefore conclude that the ORF7b protein is not only an accessory protein but a structural component of the SARS-CoV virion.

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