In vitro protein translocation across the yeast endoplasmic reticulum: ATP-dependent post-translational translocation of the prepro-$alpha;-factor

Cell ◽  
1986 ◽  
Vol 45 (3) ◽  
pp. 397-406 ◽  
Author(s):  
W HANSEN
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Translocation ◽  
Alpha Factor ◽  
Vitro Protein

scholarly journals SecA Dimer Cross-Linked at Its Subunit Interface Is Functional for Protein Translocation

2006 ◽  
Vol 188 (1) ◽  
pp. 335-338 ◽  
Author(s):  
Lucia B. Jilaveanu ◽  
Donald Oliver
Keyword(s):  
Conformational Changes ◽  
Protein Transport ◽  
Protein Translocation ◽  
Directed Mutagenesis ◽  
Subunit Interface ◽  
Cargo Proteins ◽  
Considerable Controversy ◽  
The Subject ◽  
Vitro Protein

ABSTRACT SecA facilitates protein transport across the eubacterial plasma membrane by its association with cargo proteins and the SecYEG translocon, followed by ATP-driven conformational changes that promote protein translocation in a stepwise manner. Whether SecA functions as a monomer or a dimer during this process has been the subject of considerable controversy. Here we utilize cysteine-directed mutagenesis along with the crystal structure of the SecA dimer to create a cross-linked dimer at its subunit interface, which was normally active for in vitro protein translocation.

scholarly journals Genetic Interactions betweenKAR7/SEC71,KAR8/JEM1,KAR5, andKAR2during Nuclear Fusion inSaccharomyces cerevisiae

1999 ◽  
Vol 10 (3) ◽  
pp. 609-626 ◽  
Author(s):  
Valeria Brizzio ◽  
Waheeda Khalfan ◽  
Don Huddler ◽  
Christopher T. Beh ◽  
Søren S.L. Andersen ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Interactions ◽  
Elevated Temperatures ◽  
Protein Translocation ◽  
Nuclear Fusion ◽  
Genetic Interactions ◽  
Dnaj Protein ◽  
Electron Microscopy Analysis ◽  
Chaperone Complex

During mating of Saccharomyces cerevisiae, two nuclei fuse to produce a single diploid nucleus. Two genes,KAR7 and KAR8, were previously identified by mutations that cause defects in nuclear membrane fusion.KAR7 is allelic to SEC71, a gene involved in protein translocation into the endoplasmic reticulum. Two other translocation mutants, sec63-1 andsec72Δ, also exhibited moderate karyogamy defects. Membranes from kar7/sec71Δ andsec72Δ, but not sec63-1, exhibited reduced membrane fusion in vitro, but only at elevated temperatures. Genetic interactions between kar7 andkar5 mutations were suggestive of protein–protein interactions. Moreover, in sec71 mutants, Kar5p was absent from the SPB and was not detected by Western blot or immunoprecipitation of pulse-labeled protein. KAR8 is allelic to JEMI, encoding an endoplasmic reticulum resident DnaJ protein required for nuclear fusion. Overexpression of KAR8/JEM1 (but notSEC63) strongly suppressed the mating defect ofkar2-1, suggesting that Kar2p interacts with Kar8/Jem1p for nuclear fusion. Electron microscopy analysis of kar8mutant zygotes revealed a nuclear fusion defect different fromkar2, kar5, and kar7/sec71mutants. Analysis of double mutants suggested that Kar5p acts before Kar8/Jem1p. We propose the existence of a nuclear envelope fusion chaperone complex in which Kar2p, Kar5p, and Kar8/Jem1p are key components and Sec71p and Sec72p play auxiliary roles.

scholarly journals Functional interaction of cytosolic hsp70 and a DnaJ-related protein, Ydj1p, in protein translocation in vivo.

1996 ◽  
Vol 16 (8) ◽  
pp. 4378-4386 ◽  
Author(s):  
J Becker ◽  
W Walter ◽  
W Yan ◽  
E A Craig
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Translocation ◽  
Genetic Interaction ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Alpha Factor ◽  
Synthetically Lethal ◽  
Shock Proteins ◽  
Related Proteins

In order to analyze the in vivo role of the SSA class of cytosolic 70-kDa heat shock proteins (hsps) of Saccharomyces cerevisiae, we isolated a temperature-sensitive mutant of SSA1. The effect of a shift of mutant cells (ssa1ts ssa2 ssa3 ssa4) from the permissive temperature of 23 degrees C to the nonpermissive temperature of 37 degrees C on the processing of several precursor proteins translocated into the endoplasmic reticulum or mitochondria was assessed. Of three mitochondrial proteins tested, the processing of only one, the beta subunit of the F1F0 ATPase, was dramatically affected. Of six proteins destined for the endoplasmic reticulum, the translocation of only prepro-alpha-factor and proteinase A was inhibited. The processing of prepro-alpha-factor was inhibited within 2 min of the shift to 37 degrees C, suggesting a direct effect of the hsp70 defect on translocation. More than 50% of radiolabeled alpha-factor accumulated in the precursor form, with the remainder rapidly reaching the mature form. However, the translocation block was complete, as the precursor form could not be chased through the translocation pathway. Since DnaJ-related proteins are known to interact with hsp70s and strains containing conditional mutations in a dnaJ-related gene, YDJ1, are defective in translocation of prepro-alpha-factor, we looked for a genetic interaction between SSA genes and YDJ1 in vivo. We found that a deletion mutation of YDJ1 was synthetically lethal in a ssa1ts ssa2 ssa3 ssa4 background. In addition, a strain containing a single functional SSA gene, SSA1, and a deletion of YDJ1 accumulated the precursor form of alpha-factor. However, no genetic interaction was observed between a YDJ1 mutation and mutations in the SSB genes, which encode a second class of cytosolic hsp70 chaperones. These results are consistent with SSA proteins and Ydj1p acting together in the translocation process.

scholarly journals Secretory protein translocation in a yeast cell-free system can occur posttranslationally and requires ATP hydrolysis.

1986 ◽  
Vol 102 (5) ◽  
pp. 1543-1550 ◽  
Author(s):  
M G Waters ◽  
G Blobel
Keyword(s):  
Atp Hydrolysis ◽  
Protein Translocation ◽  
Secretory Protein ◽  
Translation System ◽  
Yeast Saccharomyces Cerevisiae ◽  
Free System ◽  
In Vitro System ◽  
Alpha Factor ◽  
Microsomal Membranes

We describe an in vitro system with all components derived from the yeast Saccharomyces cerevisiae that can translocate a yeast secretory protein across microsomal membranes. In vitro transcribed prepro-alpha-factor mRNA served to program a membrane-depleted yeast translation system. Translocation and core glycosylation of prepro-alpha-factor were observed when yeast microsomal membranes were added during or after translation. A membrane potential is not required for translocation. However, ATP is required for translocation and nonhydrolyzable analogues of ATP cannot serve as a substitute. These findings suggest that ATP hydrolysis may supply the energy required for translocation of proteins across the endoplasmic reticulum.

scholarly journals Sac1p mediates the adenosine triphosphate transport into yeast endoplasmic reticulum that is required for protein translocation.

1995 ◽  
Vol 131 (6) ◽  
pp. 1377-1386 ◽  
Author(s):  
P Mayinger ◽  
V A Bankaitis ◽  
D I Meyer
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Translocation ◽  
Protein A ◽  
Lipid Vesicles ◽  
Secretory Proteins ◽  
Transport Activity ◽  
Vitro Assay ◽  
Microsomal Membranes

Protein translocation into the yeast endoplasmic reticulum requires the transport of ATP into the lumen of this organelle. Microsomal ATP transport activity was reconstituted into proteoliposomes to characterize and identify the transporter protein. A polypeptide was purified whose partial amino acid sequence demonstrated its identity to the product of the SAC1 gene. Accordingly, microsomal membranes isolated from strains harboring a deletion in the SAC1 gene (sac1 delta) were found to be deficient in ATP-transporting activity as well as severely compromised in their ability to translocate nascent prepro-alpha-factor and preprocarboxypeptidase Y. Proteins isolated from the microsomal membranes of a sac1 delta strain were incapable of stimulating ATP transport when reconstituted into the in vitro assay system. When immunopurified to homogeneity and incorporated into artificial lipid vesicles, Sac1p was shown to reconstitute ATP transport activity. Consistent with the requirement for ATP in the lumen of the ER to achieve the correct folding of secretory proteins, the sac1 delta strain was shown to have a severe defect in transport of procarboxypeptidase Y out of the ER and into the Golgi complex in vivo. The collective data indicate an intimate role for Sac1p in the transport of ATP into the ER lumen.

scholarly journals Sls1p Stimulates Sec63p-Mediated Activation of Kar2p in a Conformation-Dependent Manner in the Yeast Endoplasmic Reticulum

2000 ◽  
Vol 20 (18) ◽  
pp. 6923-6934 ◽  
Author(s):  
Mehdi Kabani ◽  
Jean-Marie Beckerich ◽  
Claude Gaillardin
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Translocation ◽  
Synthetic Lethality ◽  
In Vitro Assays ◽  
Endoplasmic Reticulum Membrane ◽  
Dependent Manner ◽  
Dose Dependent

ABSTRACT We previously characterized the SLS1 gene in the yeastYarrowia lipolytica and showed that it interacts physically with YlKar2p to promote translocation across the endoplasmic-reticulum membrane (A. Boisramé, M. Kabani, J. M. Beckerich, E. Hartmann, and C. Gaillardin, J. Biol. Chem. 273:30903–30908, 1998). A Y. lipolytica Kar2p mutant was isolated that restored interaction with an Sls1p mutant, suggesting that the interaction with Sls1p could be nucleotide and/or conformation dependent. This result was used as a working hypothesis for more accurate investigations in Saccharomyces cerevisiae. We show by two-hybrid an in vitro assays that the S. cerevisiae homologue of Sls1p interacts with ScKar2p. Using dominant lethal mutants of ScKar2p, we were able to show that ScSls1p preferentially interacts with the ADP-bound conformation of the molecular chaperone. Synthetic lethality was observed between ΔScsls1 and translocation-deficientkar2 or sec63-1 mutants, providing in vivo evidence for a role of ScSls1p in protein translocation. Synthetic lethality was also observed with ER-associated degradation and folding-deficient kar2 mutants, strongly suggesting that Sls1p functions are not restricted to the translocation process. We show that Sls1p stimulates in a dose-dependent manner the binding ofScKar2p on the lumenal J domain of Sec63p fused to glutathione S-transferase. Moreover, Sls1p is shown to promote the Sec63p-mediated activation of Kar2p's ATPase activity. Our data strongly suggest that Sls1p could be the first GrpE-like protein described in the endoplasmic reticulum.

scholarly journals SEC62 encodes a putative membrane protein required for protein translocation into the yeast endoplasmic reticulum.

1989 ◽  
Vol 109 (6) ◽  
pp. 2653-2664 ◽  
Author(s):  
R J Deshaies ◽  
R Schekman
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Translocation ◽  
Signal Sequence ◽  
Alpha Helix ◽  
Wild Type ◽  
Cytosol Fraction ◽  
Arginine Residues ◽  
Mutant Cells

Yeast sec62 mutant cells are defective in the translocation of several secretory precursor proteins into the lumen of the endoplasmic reticulum (Rothblatt et al., 1989). The deficiency, which is most restrictive for alpha-factor precursor (pp alpha F) and preprocarboxypeptidase Y, has been reproduced in vitro. Membranes isolated from mutant cells display low and labile translocation activity with pp alpha F translated in a wild-type cytosol fraction. The defect is unique to the membrane fraction because cytosol from mutant cells supports translocation into membranes from wild-type yeast. Invertase assembly is only partly affected by the sec62 mutation in vivo and is nearly normal with mutant membranes in vitro. A potential membrane location for the SEC62 gene product is supported by evaluation of the molecular clone. DNA sequence analysis reveals a 32-kD protein with no obvious NH2-terminal signal sequence but with two domains of sufficient length and hydrophobicity to span a lipid bilayer. Sec62p is predicted to display significant NH2- and COOH-terminal hydrophilic domains on the cytoplasmic surface of the ER membrane. The last 30 amino acids of the COOH terminus may form an alpha-helix with 14 lysine and arginine residues arranged uniformly about the helix. This domain may allow Sec62p to interact with other proteins of the putative translocation complex.

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