Abstract
Heat shock proteins of 70 kDa (Hsp70s) and their J domain-containing Hsp40 cofactors are highly conserved chaperone pairs that facilitate a large number of cellular processes. The observation that each Hsp70 partners with many J domain-containing proteins (JDPs) has led to the hypothesis that Hsp70 function is dictated by cognate JDPs. If this is true, one might expect highly divergent Hsp70-JDP pairs to be unable to function in vivo. However, we discovered that, when a yeast cytosolic JDP, Ydj1, was targeted to the mammalian endoplasmic reticulum (ER), it interacted with the ER-lumenal Hsp70, BiP, and bound to BiP substrates. Conversely, when a mammalian ER-lumenal JDP, ERdj3, was directed to the yeast cytosol, it rescued the temperature-sensitive growth phenotype of yeast-containing mutant alleles in two cytosolic JDPs, HLJ1 and YDJ1, and activated the ATP hydrolysis rate of Ssa1, the yeast cytosolic Hsp70 that partners with Hlj1 and Ydj1. Surprisingly, ERdj3 mutants that were compromised for substrate binding were unable to rescue the hlj1ydj1 growth defect even though they stimulated the ATPase activity of Ssa1. Yet, J domain mutants of ERdj3 that were defective for interaction with Ssa1 restored the growth of hlj1ydj1 yeast. Taken together, these data suggest that the substrate binding properties of certain JDPs, not simply the formation of unique Hsp70-JDP pairs, are critical to specify in vivo function.
Highlights
Complexes in a nucleotide-dependent manner, these chaperones play critical roles in diverse cellular processes [1, 2]
The J domain-containing proteins (JDPs) can be classified into three groups [11, 12]: (i) type I JDPs are most similar to DnaJ and contain a J domain followed by a glycine/phenylalanine-rich region and a cysteine-rich region with four repeats of a CXXCXGXG-type zinc finger; (ii) type II JDPs lack the cysteine-rich region and are unable to coordinate Zn2ϩ; and (iii) type III JDPs only have the J domain in common with DnaJ
A single Hsp70 can interact with different JDPs to form unique Hsp70-JDP pairs that participate in specific cellular functions [12, 13]
Summary
Preparation of Ydj, Hlj, ERdj, and ERdj Constructs—To target Ydj to the mammalian ER, a signal sequence (ss) was engineered onto the N terminus of Ydj using pAV4 [18] as a template and the following PCR primers (lowercase letters represent the inserted signal sequence, and the underlined letters indicate a BamHI site): 5Ј primer: CGGGATCCatggctccgcagaacctgagcaccttttgcctgttgctgctatacctcatcggggcggtgattgccGTTAAAGAAACTAAGTTTTACGATATTCTAGGTGTTCC, and 3Ј primer: CGGGATCCTCATTGAGATGCACATTGAACACCTTC. A cytosolic form of ERdj was constructed by amplifying ERdj with the primer pair (the underlined letters on the 5Ј primer represent the beginning of the coding sequence for the cytosolic form of ERdj protein without its signal sequence, while the underlined sequence on the 3Ј primer represents the farnesylation sequence; the lowercase letters represent the restriction sites used for cloning): 5Ј primer: CGgaatccTTAATTCTGGCCTCAAAAAGCTACTATGATATCTT, and 3Ј primer: GGaagcttTCATTTGAGATGCACACTGTCCTGAACAGTCAGTGTATGTAGTAA, digested with the EcoRI and HindIII enzymes, and inserted into the pGPD426 vector. Yeast—Wild-type cells transformed with an empty vector or plasmids expressing ERdj, ERdj3-CaaX, ERdj, or ERdj4-CaaX were grown to logarithmic phase (A600 of 0.8 – 1.0) in selective synthetic complete medium containing 2% glucose. The molar amounts of Hsp70s and JDPs present in the reaction are indicated in the figure legends
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