PMC:2195774 / 17993-21832 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":"YYS Fusion Proteins Partially Rescue Hap1 Defect In Vivo\nSis1 and Ydj1 have important functions in the cell, but few of their specific in vivo roles are known. Little is known about Sis1 function, although it has been reported to be required for the initiation of translation (Zhong and Arndt 1993). Ydj1 cooperates with the Hsp70 Ssa in the translocation of preproteins into the ER and mitochondria (Caplan et al. 1992a; Becker et al. 1996). In addition, Ydj1 has been shown to be required for the in vivo maturation of the heterologous proteins v-src and steroid receptors via the Hsp90 pathway (Kimura et al. 1995; Fliss et al. 1999; Johnson and Craig 2000), but native substrates have been elusive. However, recent evidence suggests that the transcription factor Hap1, which regulates the expression of CYC1 and other genes encoding respiratory functions in a heme-dependent manner (Zitomer and Lowry 1992), is such a substrate. When heme levels are low, Ydj1 and the yeast Hsp90, Hsp82, bind Hap1, and Hap1 activity is low. When heme levels increase, Ydj1 and Hsp82 dissociate from Hap1, and its transcriptional activity increases. Hsp82 is believed to play a role in the activation of Hap1, as decreased levels of Hsp82 led to a large decrease in the overall activity of a Hap1 reporter gene construct without affecting Hap1 levels (Zhang et al. 1998).\nTo determine whether Ydj1 also plays a role in the activation of Hap1, we examined Hap1 activity in ydj1 mutant strains containing a hem1 mutation, which allowed manipulation of heme levels. Varying amounts of the heme precursor, ALA, were added to the media to maintain high or low levels of heme. Hap1 activity was monitored by measuring transcription from the UAS1 of the CYC1 promoter linked to the lacZ gene (Guarente et al. 1984). In low levels of ALA (4 μg/ml), strains expressing mutant or wild-type YDJ1 had similarly low levels of β-galactosidase activity, 1–4 U (data not shown). In the presence of high concentrations of ALA (250 μg/ml), cells expressing wild-type YDJ1 had dramatically higher activity, 343 U. In contrast, cells expressing the J plus G/F region of Ydj1 (ydj1-104) had only 30 U of activity (Fig. 5 A), indicating that deletion of the substrate-binding region of Ydj1 causes an activation defect of Hap1, resulting in \u003e10% of the activity observed in wild-type cells.\nSince the substrate-binding region of Ydj1 is required for activity of Hap1, we were able to directly test whether the substrate-binding region of Sis1 is able to replace Ydj1 in this function. For this, we used fusion proteins that join the J plus G/F of Ydj1 to the G/M region and domain I of Sis1 (Fig. 5 A). In the absence of ydj1, expression of SIS1 from a high copy plasmid restored wild-type levels of Hap1 activity. In addition, expression of YYS-262 partially restored Hap1 activity (180 β-galactosidase U), whereas cells expressing YYS-206 exhibited the same level of Hap1 activity as ydj1-104. Thus, YYS-262 is able to substantially rescue a specific defect, that is loss of Hap1 activity that arises in the absence of the substrate-binding region of Ydj1.\nTo determine if YYS-262 could more generally carry out Ydj1 functions, we tested its effect on growth of a ydj1 strain at 30°C and 34°C. As previously reported, the ydj1-104 construct partially rescues growth at 30°C but exhibits a severe temperature-sensitive phenotype (Johnson and Craig 2000). YYS-262 allowed near wild-type levels of growth at 30°C and 34°C (Fig. 5 A), but not 37°C (data not shown). YYS-206 could not support growth at 34°C, supporting evidence from Fig. 2 that this truncation of domain I is not fully functional. Thus, in addition to rescuing the Hap1 defect, domain I of the Sis1 COOH terminus can rescue the growth defect that arises in the absence of the substrate-binding region of Ydj1.","divisions":[{"label":"Title","span":{"begin":0,"end":56}}],"tracks":[]}