[46] In vitro protein translocation across microsomal membranes of Saccharomyces cerevisiae

1991 ◽  
pp. 675-682 ◽  
Author(s):  
Pablo D. Garcia ◽  
William Hansen ◽  
Peter Walter
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Translocation ◽  
Microsomal Membranes ◽  
Vitro Protein

scholarly journals The alpha subunit of the Saccharomyces cerevisiae oligosaccharyltransferase complex is essential for vegetative growth of yeast and is homologous to mammalian ribophorin I.

1995 ◽  
Vol 128 (4) ◽  
pp. 525-536 ◽  
Author(s):  
S Silberstein ◽  
P G Collins ◽  
D J Kelleher ◽  
P J Rapiejko ◽  
R Gilmore
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Vegetative Growth ◽  
Functional Characterization ◽  
Glycosylation Site ◽  
Alpha Subunit ◽  
Protein Sequence Analysis ◽  
Exogenous Lipid ◽  
Microsomal Membranes ◽  
High Mannose

Oligosaccharyltransferase mediates the transfer of a preassembled high mannose oligosaccharide from a lipid-linked oligosaccharide donor to consensus glycosylation acceptor sites in newly synthesized proteins in the lumen of the rough endoplasmic reticulum. The Saccharomyces cerevisiae oligosaccharyltransferase is an oligomeric complex composed of six nonidentical subunits (alpha-zeta), two of which are glycoproteins (alpha and beta). The beta and delta subunits of the oligosaccharyltransferase are encoded by the WBP1 and SWP1 genes. Here we describe the functional characterization of the OST1 gene that encodes the alpha subunit of the oligosaccharyltransferase. Protein sequence analysis revealed a significant sequence identity between the Saccharomyces cerevisiae Ost1 protein and ribophorin I, a previously identified subunit of the mammalian oligosaccharyltransferase. A disruption of the OST1 locus was not tolerated in haploid yeast showing that expression of the Ost1 protein is essential for vegetative growth of yeast. An analysis of a series of conditional ost1 mutants demonstrated that defects in the Ost1 protein cause pleiotropic underglycosylation of soluble and membrane-bound glycoproteins at both the permissive and restrictive growth temperatures. Microsomal membranes isolated from ost1 mutant yeast showed marked reductions in the in vitro transfer of high mannose oligosaccharide from exogenous lipid-linked oligosaccharide to a glycosylation site acceptor tripeptide. Microsomal membranes isolated from the ost1 mutants contained elevated amounts of the Kar2 stress-response 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 Protein translocation across the yeast microsomal membrane is stimulated by a soluble factor.

1986 ◽  
Vol 103 (6) ◽  
pp. 2629-2636 ◽  
Author(s):  
M G Waters ◽  
W J Chirico ◽  
G Blobel
Keyword(s):  
Heat Treatment ◽  
Saccharomyces Cerevisiae ◽  
Protein Translocation ◽  
Sedimentation Coefficient ◽  
Protein S ◽  
Microsomal Membrane ◽  
Supernatant Fraction ◽  
Soluble Factor ◽  
Microsomal Membranes ◽  
Stimulation Of

We have found that a soluble activity present in the postribosomal supernatant fraction of Saccharomyces cerevisiae stimulates posttranslational translocation of yeast prepro-alpha-factor across yeast microsomal membranes. Stimulation of translocation is not due to a nonspecific affect on ATP levels. The activity is likely to be due to protein(s) as it is destroyed by N-ethylmaleimide, protease, or heat treatment but not by incubation with RNase. Its apparent sedimentation coefficient is approximately 9.6 S.

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 Ribonuclease-neutralized pancreatic microsomal membranes from livestock for in vitro co-translational protein translocation

2015 ◽  
Vol 484 ◽  
pp. 102-104 ◽  
Author(s):  
Kurt Vermeire ◽  
Susanne Allan ◽  
Becky Provinciael ◽  
Enno Hartmann ◽  
Kai-Uwe Kalies
Keyword(s):  
Protein Translocation ◽  
Microsomal Membranes

scholarly journals Interaction between BiP and Sec63p is required for the completion of protein translocation into the ER of Saccharomyces cerevisiae.

1995 ◽  
Vol 131 (5) ◽  
pp. 1163-1171 ◽  
Author(s):  
S K Lyman ◽  
R Schekman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Translocation ◽  
Essential Genes ◽  
Precursor Protein ◽  
Conserved Residue ◽  
Er Membrane

To clarify the roles of Kar2p (BiP) and Sec63p in translocation across the ER membrane in Saccharomyces cerevisiae, we have utilized mutant alleles of the essential genes that encode these proteins: kar2-203 and sec63-1. Sanders et al. (Sanders, S. L., K. M. Whitfield, J. P. Vogel, M. D. Rose, and R. W. Schekman. 1992. Cell. 69:353-365) showed that the translocation defect of the kar2-203 mutant lies in the inability of the precursor protein to complete its transit across the membrane, suggesting that the lumenal hsp70 homologue Kar2p (BiP) binds the transiting polypeptide in order to facilitate its passage through the pore. We now show that mutation of a conserved residue (A181-->T) (Nelson, M. K., T. Kurihara, and P. Silver. 1993. Genetics. 134:159-173) in the lumenal DnaJ box of Sec63p (sec63-1) results in an in vitro phenotype that mimics the precursor stalling defect of kar2-203. We demonstrate by several criteria that this phenotype results specifically from a defect in the lumenal interaction between Sec63p and BiP: Neither a sec62-1 mutant nor a mutation in the cytosolically exposed domain of Sec63p causes precursor stalling, and interaction of the sec63-1 mutant with the membranebound components of the translocation apparatus is unimpaired. Additionally, dominant KAR2 suppressors of sec63-1 partially relieve the stalling defect. Thus, proper interaction between BiP and Sec63p is necessary to allow the precursor polypeptide to complete its transit across the membrane.

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