In all organisms studied, heat shock or other stressful stimuli lead to profound changes in gene expression. During heat shock, a small group of evolutionarily conserved proteins, termed heat shock proteins (hsps), are synthesized in abunDance; Several hsps are known to have vital cellular function even at normal temperature. These hsps have the capacity to bind polypeptide chains and have a probable role in the folding, translocation, and assembly pathways of many proteins. One such protein, hsp60, is located within mitochondria, is highly conserved in evolution, and based on immunological and physical characterization, is a member of a family of proteins that includes chloroplast chaperonins and bacterial proteins such as the groEL protein of E. coli that functions in assembly of bacteriophages. In order to characterize further the hsp60 gene family, the yeast nuclear gene which codes for this protein was cloned and sequenced. Sequence analysis confirmed that HSP60 is part of a conserved gene family. The deduced amino acid sequence of hsp60 is 54% and 43% identical to those for groEL and the ribuliouse bisthosphate carboxylase submits binding protein, respectively, assuming several small additives or deletions;The results of an experiment in which a restriction fragment containing the HIS3 gene was cloned into an internal restriction site within HSP60 demonstrated that HSP60 is essential in yeast: Cells harboring such an interrupted gene were not viable at either normal or elevated temperature. Genetic analysis also revealed that HSP60 resides at the same genetic locus as the defective gene in a temperature sensitive yeast mutant in which polypeptides are normally imported and proteolytically processed within mitochondria, but not assembled into holoenzymes. Thus, hsp60 is required for assembly of holoenzymes within mitochondria;Further experiments characterized the regulation of HSP60, especially with respect to conditions that likely affect macromolecular assembly within mitochondria. Western and Northern blotting analyses revealed that HSP60 expression is enhanced in cells that lack mitochondrial DNA, indicating a signal pathway between mitochondria and nucleus. This mitochondrial genotype effect was additive with the increased expression due to heat shock. In addition, a strong, early induction of HSP60 following release from glucose repression was observed. The inductions due to deletion of mitochondrial DNA, carbon source, and heat shock utilized the same transcription initiation site and were mediated by sequence information within 400 base pairs of this site.