PMC:2195774 / 9377-12163
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{"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 Region in the COOH Terminus of Sis1\nSubstrate binding is likely the essential function of the COOH terminus of Sis1, as a proteolytic fragment encompassing a.a. 171–352 is capable of binding denatured substrates in vitro (Lu and Cyr 1998b). Recently, the crystal structure of this fragment revealed three domains (Fig. 2 C). Domains I and II have similar folds. However, domain I contains a prominent hydrophobic depression and thus was proposed to contain the substrate-binding site. In addition, the extreme COOH terminus (a.a. 338–352) is a dimerization domain (Sha et al. 2000).\nTo determine which sequences in the COOH-terminal fragment of Sis1 are essential in the absence of the COOH terminus of Ydj1, SIS1 truncation mutants were transformed into the sis1 ydj1 strain expressing ydj1-134. When transformants were spotted onto plates containing 5-FOA, colonies appeared in strains expressing sis1-206, -253, -262, -297, and wild type (Fig. 2 A). However, when cells picked from these colonies were tested more carefully (Fig. 2 B, upper panel), it became apparent that cells expressing Sis1-206 grew slowly at 30°C, even though this truncation is expressed at wild-type levels (Fig. 2 B, lower panel). Cells expressing Sis1-253, -262, or -297 exhibited wild-type growth rates.\nThese results clearly indicate that domain II (a.a. 260–336) and the dimerization region (a.a. 338–352) of Sis1 are not required for normal growth at 30°C even in the absence of the COOH terminus of Ydj1. However, domain I (a.a. 180–255) is required, as sis1-206 cells grow very slowly, and sis1-171 cells are inviable in this assay. Sha et al. 2000 proposed that, within domain I, residues V184 and L186 from β strand 1, F201, and I203 from β strand 2, and F251 from β strand 5 interact with bound substrate (Fig. 2 C), however, the role of these residues in substrate interaction has not yet been established. Sis1-262 and Sis1-253 contain these residues, whereas Sis1-206 truncates after β strand 2 and thus lacks F251 and β sheets that are probably important in stabilizing the domain. Our results suggest that Sis1-262 contains a functional substrate-binding domain. However, at this time, the only available in vitro assay of Sis1 chaperone function measures the ability to bind and refold denatured luciferase in cooperation with Hsp70 (Sha et al. 2000). Since activity in these in vitro assays is dependent on the presence of the dimerization domain of Sis1, we are unable to monitor the in vitro substrate-binding activity of Sis1-262 in the absence of domain II and the dimerization domain. However, our genetic results demonstrate a critical role of domain I in the absence of the COOH terminus of Ydj1 and are consistent with that role being substrate binding.","divisions":[{"label":"Title","span":{"begin":0,"end":65}}],"tracks":[]}