PMC:2195774 / 12165-16214 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/2195774","sourcedb":"PMC","sourceid":"2195774","source_url":"http://www.ncbi.nlm.nih.gov/pmc/2195774","text":"Localization of the Essential Regions in the COOH Terminus of Ydj1\nHaving found the region of Sis1 that is critical in the absence of the COOH terminus of Ydj1, we asked the same question of Ydj1: what portion of Ydj1 is essential in the absence of the COOH terminus of Sis1? The substrate-binding region of Ydj1 has been localized between a.a. 179 and 384 of Ydj1 (Lu and Cyr 1998a), a fragment that contains part of the cysteine-rich region and most of the COOH terminus (Fig. 1 A). The extreme COOH terminus of Ydj1 contains the CAAX box, the site of a farnesylation modification thought to be involved in localization of Ydj1 to membranes (Caplan et al. 1992b). Truncation and point mutants of YDJ1, picked because they disrupt function of these regions, were transformed into the sis1 ydj1 strain that carried wild-type SIS1 on a URA3-based plasmid and also expressed sis1-121. The shortest truncation that allowed growth in the absence of full-length Sis1 was ydj1-274, which contains not only the J plus G/F region, but also the cysteine-rich region and 68 a.a. of the COOH terminus (Fig. 3 A). When retested for growth at 30°C, it was clear that ydj1-274 allowed cells to grow nearly as well as wild-type YDJ1.\nThese results are consistent with substrate binding being a critical role of the region distal to the J plus G/F. Mutant DnaJ proteins having alterations in the cysteine-rich region have shown defects in suppression of aggregation of rhodanese and binding to some, but not all, protein substrates (Banecki et al. 1996; Szabo et al. 1996). Therefore, the cysteine-rich region is implicated in binding of at least some protein substrates. However, a fragment containing only the J plus G/F plus cysteine-rich region of Ydj1 (ydj1-206) did not support viability when sis1-121 was the only copy of SIS1 present. This result suggests that, in addition to the cysteine-rich region, the first 68 a.a. of the COOH terminus are required for the ability to bind substrates in vivo. Thus, our results are in agreement with studies in which a fragment of Ydj1 encompassing a.a. 179–384 bound denatured substrate (Lu and Cyr 1998a).\nOther mutants that could be recovered on 5-FOA did not grow as well as ydj1-274 (Fig. 3 B). Although sequences required for substrate binding or other sequences important in allowing proper transient interactions with substrates have not been clearly established, the amino acid substitution G315D led to in vivo defects in binding of glucocorticoid receptor (Kimura et al. 1995). In vitro, G315D was found to be defective both in suppressing rhodanase aggregation and refolding denatured luciferase (Lu and Cyr 1998a). Consistent with a role in substrate interaction, an amino acid substitution at the same residue, ydj1-G315E, exhibited severe growth defects when sis1-121 was the only SIS1 gene present (Fig. 3 B). Although the most straightforward interpretation of these results is that G315 and surrounding residues are important for substrate binding, it is also possible that, in light of the robust growth of ydj1-274, COOH-terminal sequences can act negatively on more proximal residues, inhibiting their ability to function. Consistent with this idea, the truncation mutant ydj1-363, which results in removal of fewer residues from the COOH terminus than ydj1-274, and ydj1-C406S, which alters the farnesylation signal, grow poorly in the presence of sis1-121 compared with the truncation ydj1-274. The C406S mutation has been shown to result in a decrease in the membrane association of Ydj1 (Caplan et al. 1992b). The associated temperature-sensitive growth defect was attributed to this alteration in cellular localization. However, in other proteins, the farnesylation modification has been shown to not only affect cellular localization but also protein–protein interactions (Sinensky 2000); so it is possible that farnesylation has additional effects on Ydj1 function. Further studies will be required to determine how the farnesylation modification of Ydj1 affects in vivo chaperone 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