IDENTIFICATION OF IN-VIVO SUBSTRATES OF THE YEAST MITOCHONDRIAL CHAPERONINS REVEALS OVERLAPPING BUT NONIDENTICAL REQUIREMENT FOR HSP60 AND HSP10

The mechanism of chaperonin-assisted protein folding has been mostly a nalyzed in vitro using non-homologous substrate proteins. In order to understand the relative importance of hsp60 and hsp10 in the living ce ll, homologous substrate proteins need to be identified and analyzed. We have devised a novel screen to test the folding of a large variety of homologous substrates in the mitochondrial matrix in the absence or presence of functional hsp60 or hsp10, The identified substrates have an M-r of 15-90 kDa and fall into three groups: (i) proteins that req uire both hsp60 and hsp10 for correct folding; (ii) proteins that comp letely fail to fold after inactivation of hsp60 but are unaffected by the inactivation of hsp10; and (iii) newly imported hsp60 itself, whic h is more severely affected by inactivation of hsp10 than by inactivat ion of pre-existing hsp60, The majority of the identified substrates a re group I proteins. For these, the lack of hsp60 function has a more pronounced effect than inactivation of hsp10, We suggest that homologo us substrate proteins have differential chaperonin requirements, indic ating that hsp60 and hsp10 do not always act as a single functional un it in vivo.