Lu Z, Cyr D M
Department of Cell Biology, School of Medicine, University of Alabama Medical Center, Birmingham, Alabama 35294-0005, USA.
J Biol Chem. 1998 Mar 6;273(10):5970-8. doi: 10.1074/jbc.273.10.5970.
Ydj1 is a member of the Hsp40 (DnaJ-related) chaperone family that facilitates cellular protein folding by regulating Hsp70 ATPase activity and binding unfolded polypeptides. Ydj1 contains four conserved subdomains that appear to represent functional units. To define the action of these regions, protease-resistant Ydj1 fragments and Ydj1 mutants were analyzed for activities exhibited by the unmodified protein. The Ydj1 mutant proteins analyzed were unable to support growth of yeast at elevated temperatures and were found to have alterations in the J-domain (Ydj1 H34Q), zinc finger-like region (Ydj1 C159T), and conserved carboxyl terminus (Ydj1 G315D). Fragment Ydj1 (1-90) contains the J-domain and a small portion of the G/F-rich region and could regulate Hsp70 ATPase activity but could not suppress the aggregation of the model protein rhodanese. Ydj1 H34Q could not regulate the ATPase activity of Hsp70 but could bind unfolded polypeptides. The J-domain functions independently and was sufficient to regulate Hsp70 ATPase activity. Fragment Ydj1 (179-384) could suppress rhodanese aggregation but was unable to regulate Hsp70. Ydj1 (179-384) contains the conserved carboxyl terminus of DnaJ but is missing the J-domain, G/F-rich region, and a major portion of the zinc finger-like region. Ydj1 G315D exhibited severe defects in its ability to suppress rhodanese aggregation and form complexes with unfolded luciferase. The conserved carboxyl terminus of Ydj1 appeared to participate in the binding of unfolded polypeptides. Ydj1 C159T could form stable complexes with unfolded proteins and suppress protein aggregation but was inefficient at refolding denatured luciferase. The zinc finger-like region of Ydj1 appeared to function in conjunction with the conserved carboxyl terminus to fold proteins. However, Ydj1 does not require an intact zinc finger-like region to bind unfolded polypeptides. These data suggest that the combined functions of the J-domain, zinc finger-like region, and the conserved carboxyl terminus are required for Ydj1 to cooperate with Hsp70 and facilitate protein folding in the cell.
Ydj1是热休克蛋白40（DnaJ相关）伴侣蛋白家族的成员，它通过调节热休克蛋白70（Hsp70）的ATP酶活性和结合未折叠的多肽来促进细胞内蛋白质折叠。Ydj1包含四个保守的亚结构域，似乎代表功能单元。为了确定这些区域的作用，对蛋白酶抗性的Ydj1片段和Ydj1突变体进行了分析，以检测未修饰蛋白所表现出的活性。所分析的Ydj1突变蛋白在高温下无法支持酵母生长，并且发现在J结构域（Ydj1 H34Q）、锌指样区域（Ydj1 C159T）和保守的羧基末端（Ydj1 G315D）存在改变。片段Ydj1（1 - 90）包含J结构域和一小部分富含G/F的区域，能够调节Hsp70的ATP酶活性，但不能抑制模型蛋白硫氰酸酶的聚集。Ydj1 H34Q不能调节Hsp70的ATP酶活性，但能够结合未折叠的多肽。J结构域独立发挥作用，足以调节Hsp70的ATP酶活性。片段Ydj1（179 - 384）能够抑制硫氰酸酶的聚集，但不能调节Hsp70。Ydj1（179 - 384）包含DnaJ保守的羧基末端，但缺少J结构域、富含G/F的区域和大部分锌指样区域。Ydj1 G315D在抑制硫氰酸酶聚集以及与未折叠的荧光素酶形成复合物的能力方面表现出严重缺陷。Ydj1保守的羧基末端似乎参与未折叠多肽的结合。Ydj1 C159T能够与未折叠蛋白形成稳定的复合物并抑制蛋白质聚集，但在使变性的荧光素酶复性方面效率较低。Ydj1的锌指样区域似乎与保守的羧基末端协同作用来折叠蛋白质。然而，Ydj1结合未折叠多肽并不需要完整的锌指样区域。这些数据表明，Ydj1与Hsp70协同作用并促进细胞内蛋白质折叠需要J结构域、锌指样区域和保守羧基末端的共同功能。
