The amino-terminal signal peptide on the porcine transmissible gastroenteritis coronavirus matrix protein is not an absolute requirement for membrane translocation and glycosylation

To test the hypothesis that 70-kD mitochondrial heat shock protein (mt-hsp70) has a dual role in membrane translocation of preproteins we screened preproteins in an attempt to find examples which required either only the unfoldase or only the translocase function of mt-hsp70. We found that a series of fusion proteins containing amino-terminal portions of the intermembrane space protein cytochrome b2 (cyt. b2) fused to dihydrofolate reductase (DHFR) were differentially imported into mitochondria containing mutant hsp70s. A fusion protein between the amino-terminal 167 residues of the precursor of cyt. b2 and DHFR was efficiently transported into mitochondria independently of both hsp70 functions. When the length of the cyt. b2 portion was increased and included the heme binding domain, the fusion protein became dependent on the unfoldase function of mt-hsp70, presumably caused by a conformational restriction of the heme-bound preprotein. In the absence of heme the noncovalent heme binding domain in the longer fusion proteins no longer conferred a dependence on the unfoldase function. When the cyt. b2 portion of the fusion protein was less than 167 residues, its import was still independent of mt-hsp70 function; however, deletion of the intermembrane space sorting signal resulted in preproteins that ended up in the matrix of wild-type mitochondria and whose translocation was strictly dependent on the translocase function of mt-hsp70. These findings provide strong evidence for a dual role of mt-hsp70 in membrane translocation and indicate that preproteins with an intermembrane space sorting signal can be correctly imported even in mutants with severely impaired hsp70 function.

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The Putative Amino-terminal Signal Peptide of the Cloned Rat Brain Na+-Ca2+Exchanger Gene (RBE-1) Is Not Mandatory for Functional Expression

Journal of Biological Chemistry ◽

10.1074/jbc.270.32.19120 ◽

1995 ◽

Vol 270 (32) ◽

pp. 19120-19127 ◽

Cited By ~ 22

Author(s):

Ian Furman ◽

Orna Cook ◽

Judith Kasir ◽

Walter Low ◽

Hannah Rahamimoff

Keyword(s):

Rat Brain ◽

Signal Peptide ◽

Functional Expression ◽

Amino Terminal

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The Nucleoprotein Is Required for Efficient Coronavirus Genome Replication

Journal of Virology ◽

10.1128/jvi.78.22.12683-12688.2004 ◽

2004 ◽

Vol 78 (22) ◽

pp. 12683-12688 ◽

Cited By ~ 124

Author(s):

Fernando Almazán ◽

Carmen Galán ◽

Luis Enjuanes

Keyword(s):

Heterologous Protein ◽

Cytopathic Effect ◽

Human Cell Line ◽

Genome Replication ◽

Bacterial Artificial Chromosomes ◽

Artificial Chromosomes ◽

Restriction Sites ◽

Unique Restriction ◽

Transmissible Gastroenteritis ◽

Transmissible Gastroenteritis Coronavirus

ABSTRACT The construction of a set of transmissible gastroenteritis coronavirus (TGEV)-derived replicons as bacterial artificial chromosomes is reported. These replicons were generated by sequential deletion of nonessential genes for virus replication, using a modified TGEV full-length cDNA clone containing unique restriction sites between each pair of consecutive genes. Efficient activity of TGEV replicons was associated with the presence of the nucleoprotein provided either in cis or in trans. TGEV replicons were functional in several cell lines, including the human cell line 293T, in which no or very low cytopathic effect was observed, and expressed high amounts of heterologous protein.

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Phenotypes of vesicular stomatitis virus mutants with mutations in the PSAP motif of the matrix protein

Journal of General Virology ◽

10.1099/vir.0.039800-0 ◽

2012 ◽

Vol 93 (4) ◽

pp. 857-865 ◽

Cited By ~ 16

Author(s):

Linda Obiang ◽

Hélène Raux ◽

Malika Ouldali ◽

Danielle Blondel ◽

Yves Gaudin

Keyword(s):

Vesicular Stomatitis Virus ◽

Matrix Protein ◽

Virus Assembly ◽

Susceptibility Gene ◽

Point Mutations ◽

Binding Motif ◽

Single Mutation ◽

Amino Terminal ◽

Vesicular Stomatitis ◽

Late Domains

Vesicular stomatitis virus (VSV) matrix protein (M) has a flexible amino-terminal part that recruits cellular partners. It contains a dynamin-binding site that is required for efficient virus assembly, and two motifs, 24PPPY27 and 37PSAP40, that constitute potential late domains. Late domains are present in proteins of several enveloped viruses and are involved in the ultimate step of the budding process (i.e. fission between viral and cellular membranes). In baby hamster kidney (BHK)-21 cells, it has been demonstrated that the 24PPPY27 motif binds the Nedd4 (neuronal precursor cell-expressed developmentally downregulated 4) E3 ubiquitin ligase for efficient virus budding and that the 37PSAP40 motif, although conserved among M proteins of vesiculoviruses, does not possess late-domain activity. In this study, we have re-examined the contribution of the PSAP motif to VSV budding. First, we demonstrate that VSV M indeed binds TSG101 [tumour susceptibility gene 101; a component of the ESCRT1 (endosomal sorting complex required for transport 1)] through its PSAP motif. Second, we analysed the phenotype of several recombinant mutants. We show that a double mutant with point mutations in both the PSAP and the PPPY motifs is impaired compared with a single mutant in the PPPY motif, indicating that the PSAP motif partially compensates for the lack of the PPPY motif. Mutants’ phenotypes depend on cell lines: in CERA (chicken embryo-related, Alger clone) cells, a recombinant virus with a single mutation in the PSAP motif was impaired compared with the wild type, and a mutant with a single mutation in the dynamin-binding motif was much less impaired in Vero cells than in BSR (clones of BHK-21) cells. These results have implications for the VSV budding pathway that will be discussed.

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Xylanase Attachment to the Cell Wall of the Hyperthermophilic Bacterium Thermotoga maritima

Journal of Bacteriology ◽

10.1128/jb.01149-07 ◽

2007 ◽

Vol 190 (4) ◽

pp. 1350-1358 ◽

Cited By ~ 30

Author(s):

Wolfgang Liebl ◽

Christoph Winterhalter ◽

Wolfgang Baumeister ◽

Martin Armbrecht ◽

Michael Valdez

Keyword(s):

Outer Membrane ◽

Signal Peptide ◽

Cellular Localization ◽

Culture Supernatant ◽

Thermotoga Maritima ◽

Surrounding Medium ◽

Hydrophobic Core ◽

Electron Microscopic ◽

Thin Sections ◽

Amino Terminal

ABSTRACT The cellular localization and processing of the endo-xylanases (1,4-β-d-xylan-xylanohydrolase; EC 3.2.1.8) of the hyperthermophile Thermotoga maritima were investigated, in particular with respect to the unusual outer membrane (“toga”) of this gram-negative bacterium. XynB (40 kDa) was detected in the periplasmic fraction of T. maritima cells and in the culture supernatant. XynA (120 kDa) was partially released to the surrounding medium, but most XynA remained cell associated. Immunogold labeling of thin sections revealed that cell-bound XynA was localized mainly in the outer membranes of T. maritima cells. Amino-terminal sequencing of purified membrane-bound XynA revealed processing of the signal peptide after the eighth residue, thereby leaving the hydrophobic core of the signal peptide attached to the enzyme. This mode of processing is reminiscent of type IV prepilin signal peptide cleavage. Removal of the entire XynA signal peptide was necessary for release from the cell because enzyme purified from the culture supernatant lacked 44 residues at the N terminus, including the hydrophobic part of the signal peptide. We conclude that toga association of XynA is mediated by residues 9 to 44 of the signal peptide. The biochemical and electron microscopic localization studies together with the amino-terminal processing data indicate that XynA is held at the cell surface of T. maritima via a hydrophobic peptide anchor, which is highly unusual for an outer membrane protein.

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