Interactions between Kar2p and its nucleotide exchange factors Sil1p and Lhs1p are mechanistically distinct.
Kar2p, an essential Hsp70 chaperone in the endoplasmic reticulum of Saccharomyces cerevisiae, facilitates the transport and folding of nascent polypeptides within the endoplasmic reticulum lumen. The chaperone activity of Kar2p is regulated by its intrinsic ATPase activity that can be stimulated by two different nucleotide exchange factors, namely Sil1p and Lhs1p. Here, we demonstrate that the binding requirements for Lhs1p are complex, requiring both the nucleotide binding domain plus the linker domain of Kar2p. In contrast, the IIB domain of Kar2p is sufficient for binding of Sil1p, and point mutations within IIB specifically blocked Sil1p-dependent activation while remaining competent for activation by Lhs1p. Taken together, these results demonstrate that the interactions between Kar2p and its two nucleotide exchange factors can be functionally resolved and are thus mechanistically distinct.
Hale
Sarah J
SJ
Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom.
Lovell
Simon C
SC
de Keyzer
Jeanine
J
Stirling
Colin J
CJ
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D005656:Fungal Proteins / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D005982:Glutathione Transferase / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D005656:Fungal Proteins; D018840:HSP70 Heat-Shock Proteins; C060153:KAR2 protein, yeast; C102115:LHS1 protein, S cerevisiae; D026901:Membrane Transport Proteins; C513385:SIL1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D005982:Glutathione Transferase; D000251:Adenosine Triphosphatases