Mutation of the ATP-binding pocket of SSA1 indicates that a functional interaction between Ssa1p and Ydj1p is required for post-translational translocation into the yeast endoplasmic reticulum.
The translocation of proteins across the yeast ER membrane requires ATP hydrolysis and the action of DnaK (hsp70) and DnaJ homologues. In Saccharomyces cerevisiae the cytosolic hsp70s that promote post-translational translocation are the products of the Ssa gene family. Ssa1p maintains secretory precursors in a translocation-competent state and interacts with Ydj1p, a DnaJ homologue. Although it has been proposed that Ydj1p stimulates the ATPase activity of Ssa1p to release preproteins and engineer translocation, support for this model is incomplete. To this end, mutations in the ATP-binding pocket of SSA1 were constructed and examined both in vivo and in vitro. Expression of the mutant Ssa1p's slows wild-type cell growth, is insufficient to support life in the absence of functional Ssa1p, and results in a dominant effect on post-translational translocation. The ATPase activity of the purified mutant proteins was not enhanced by Ydj1p and the mutant proteins could not bind an unfolded polypeptide substrate. Our data suggest that a productive interaction between Ssa1p and Ydj1p is required to promote protein translocation.
McClellan
A J
AJ
Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.
Brodsky
J L
JL
D000251:Adenosine Triphosphatases; D000255:Adenosine Triphosphate / Q000378:metabolism; D019943:Amino Acid Substitution; D001665:Binding Sites; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism; D005838:Genotype; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006360:Heat-Shock Proteins; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins
D005656:Fungal Proteins; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C070319:YDJ1 protein, S cerevisiae; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C095973:SSA1 protein, S cerevisiae