The glycine-phenylalanine-rich region determines the specificity of the yeast Hsp40 Sis1.
Hsp40s are ubiquitous, conserved proteins which function with molecular chaperones of the Hsp70 class. Sis1 is an essential Hsp40 of the cytosol of Saccharomyces cerevisiae, thought to be required for initiation of translation. We carried out a genetic analysis to determine the regions of Sis1 required to perform its key function(s). A C-terminal truncation of Sis1, removing 231 amino acids but retaining the N-terminal 121 amino acids encompassing the J domain and the glycine-phenylalanine-rich (G-F) region, was able to rescue the inviability of a Deltasis1 strain. The yeast cytosol contains other Hsp40s, including Ydj1. To determine which regions carried the critical determinants of Sis1 function, we constructed chimeric genes containing portions of SIS1 and YDJ1. A chimera containing the J domain of Sis1 and the G-F region of Ydj1 could not rescue the lethality of the Deltasis1 strain. However, a chimera with the J domain of Ydj1 and the G/F region of Sis1 could rescue the strain's lethality, indicating that the G-F region is a unique region required for the essential function of Sis1. However, a J domain is also required, as mutants expected to cause a disruption of the interaction of the J domain with Hsp70 are inviable. We conclude that the G-F region, previously thought only to be a linker or spacer region between the J domain and C-terminal regions of Hsp40s, is a critical determinant of Sis1 function.
Yan
W
W
Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
Craig
E A
EA
D000595:Amino Acid Sequence; D003600:Cytosol; D020043:Genes, Essential; D005800:Genes, Fungal; D005998:Glycine; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010448:Peptide Initiation Factors / Q000235:genetics / Q000378:metabolism; D010649:Phenylalanine; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D010448:Peptide Initiation Factors; D011993:Recombinant Fusion Proteins; C070320:SIS1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C070319:YDJ1 protein, S cerevisiae; D010649:Phenylalanine; D005998:Glycine