Dubaquié Y, Looser R, Rospert S
Department of Biochemistry, Biozentrum der Universität Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland.
Proc Natl Acad Sci U S A. 1997 Aug 19;94(17):9011-6. doi: 10.1073/pnas.94.17.9011.
Chaperonins are essential for the folding of proteins in bacteria, mitochondria, and chloroplasts. We have functionally characterized the yeast mitochondrial chaperonins hsp60 and hsp10. In the presence of ADP, one molecule of hsp10 binds to hsp60 with an apparent Kd of 0.9 nM and a second molecule of hsp10 binds with a Kd of 24 nM. In the presence of ATP, the purified yeast chaperonins mediate the refolding of mitochondrial malate dehydrogenase. Hsp10 inhibits the ATPase activity of hsp60 by about 40%. Hsp10(P36H) is a point mutant of hsp10 that confers temperature-sensitive growth to yeast. Consistent with the in vivo phenotype, refolding of mitochondrial malate dehydrogenase in the presence of purified hsp10(P36H) and hsp60 is reduced at 25 degrees C and abolished at 30 degrees C. The affinity of hsp10(P36H) to hsp60 as well as to Escherichia coli GroEL is reduced. However, this decrease in affinity does not correlate with the functional defect, because hsp10(P36H) fully assists the GroEL-mediated refolding of malate dehydrogenase at 30 degrees C. Refolding activity, rather, correlates with the ability of hsp10(P36H) to inhibit the ATPase of GroEL but not that of hsp60. Based on our findings, we propose that the inhibition of ATP hydrolysis is mechanistically coupled to chaperonin-mediated protein folding.
伴侣蛋白对于细菌、线粒体和叶绿体中蛋白质的折叠至关重要。我们已对酵母线粒体伴侣蛋白hsp60和hsp10进行了功能特性分析。在存在ADP的情况下，一个hsp10分子以0.9 nM的表观解离常数（Kd）与hsp60结合，第二个hsp10分子以24 nM的Kd结合。在存在ATP的情况下，纯化的酵母伴侣蛋白介导线粒体苹果酸脱氢酶的重折叠。hsp10抑制hsp60的ATP酶活性约40%。hsp10(P36H)是hsp10的一个点突变体，它使酵母具有温度敏感型生长特性。与体内表型一致，在纯化的hsp10(P36H)和hsp60存在的情况下，线粒体苹果酸脱氢酶在25℃时的重折叠减少，在30℃时则完全被抑制。hsp10(P36H)对hsp60以及对大肠杆菌GroEL的亲和力均降低。然而，这种亲和力的降低与功能缺陷并无关联，因为hsp10(P36H)在30℃时能完全协助GroEL介导的苹果酸脱氢酶重折叠。相反，重折叠活性与hsp10(P36H)抑制GroEL而非hsp60的ATP酶的能力相关。基于我们的研究结果，我们提出ATP水解的抑制在机制上与伴侣蛋白介导的蛋白质折叠相关联。
