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

Corresponding author: Jeffrey L. Brodsky, Department of Biological Sciences, University of Pittsburgh, 267 Crawford Hall, Pittsburgh, PA 15260., jbrodsky+{at}pitt.edu (E-mail)

Communicating editor: M. D. ROSE

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.

This article has been cited by other articles:

				C. M. Wright, S. W. Fewell, M. L. Sullivan, J. M. Pipas, S. C. Watkins, and J. L. Brodsky The Hsp40 Molecular Chaperone Ydj1p, Along With the Protein Kinase C Pathway, Affects Cell-Wall Integrity in the Yeast Saccharomyces cerevisiae Genetics, April 1, 2007; 175(4): 1649 - 1664. [Abstract] [Full Text] [PDF]

				X. S. Li, J. N. Sun, K. Okamoto-Shibayama, and M. Edgerton Candida albicans Cell Wall Ssa Proteins Bind and Facilitate Import of Salivary Histatin 5 Required for Toxicity J. Biol. Chem., August 11, 2006; 281(32): 22453 - 22463. [Abstract] [Full Text] [PDF]

				L. Shaner, H. Wegele, J. Buchner, and K. A. Morano The Yeast Hsp110 Sse1 Functionally Interacts with the Hsp70 Chaperones Ssa and Ssb J. Biol. Chem., December 16, 2005; 280(50): 41262 - 41269. [Abstract] [Full Text] [PDF]

				S. W. Fewell, C. M. Smith, M. A. Lyon, T. P. Dumitrescu, P. Wipf, B. W. Day, and J. L. Brodsky Small Molecule Modulators of Endogenous and Co-chaperone-stimulated Hsp70 ATPase Activity J. Biol. Chem., December 3, 2004; 279(49): 51131 - 51140. [Abstract] [Full Text] [PDF]

				R. T. Youker, P. Walsh, T. Beilharz, T. Lithgow, and J. L. Brodsky Distinct Roles for the Hsp40 and Hsp90 Molecular Chaperones during Cystic Fibrosis Transmembrane Conductance Regulator Degradation in Yeast Mol. Biol. Cell, November 1, 2004; 15(11): 4787 - 4797. [Abstract] [Full Text] [PDF]

				L. Shaner, A. Trott, J. L. Goeckeler, J. L. Brodsky, and K. A. Morano The Function of the Yeast Molecular Chaperone Sse1 Is Mechanistically Distinct from the Closely Related Hsp70 Family J. Biol. Chem., May 21, 2004; 279(21): 21992 - 22001. [Abstract] [Full Text] [PDF]

				K. Boonsirichai, J. C. Sedbrook, R. Chen, S. Gilroy, and P. H. Masson ALTERED RESPONSE TO GRAVITY Is a Peripheral Membrane Protein That Modulates Gravity-Induced Cytoplasmic Alkalinization and Lateral Auxin Transport in Plant Statocytes PLANT CELL, November 1, 2003; 15(11): 2612 - 2625. [Abstract] [Full Text] [PDF]

				H. Wegele, M. Haslbeck, J. Reinstein, and J. Buchner Sti1 Is a Novel Activator of the Ssa Proteins J. Biol. Chem., July 3, 2003; 278(28): 25970 - 25976. [Abstract] [Full Text] [PDF]

				J. Ngosuwan, N. M. Wang, K. L. Fung, and W. J. Chirico Roles of Cytosolic Hsp70 and Hsp40 Molecular Chaperones in Post-translational Translocation of Presecretory Proteins into the Endoplasmic Reticulum J. Biol. Chem., February 21, 2003; 278(9): 7034 - 7042. [Abstract] [Full Text] [PDF]

				J. L. Goeckeler, A. Stephens, P. Lee, A. J. Caplan, and J. L. Brodsky Overexpression of Yeast Hsp110 Homolog Sse1p Suppresses ydj1-151 Thermosensitivity and Restores Hsp90-dependent Activity Mol. Biol. Cell, August 1, 2002; 13(8): 2760 - 2770. [Abstract] [Full Text] [PDF]

				M. Kabani, J.-M. Beckerich, and J. L. Brodsky Nucleotide Exchange Factor for the Yeast Hsp70 Molecular Chaperone Ssa1p Mol. Cell. Biol., July 1, 2002; 22(13): 4677 - 4689. [Abstract] [Full Text] [PDF]