PURIFICATION AND BIOCHEMICAL-PROPERTIES OF SACCHAROMYCES-CEREVISIAE MDJ1P, THE MITOCHONDRIAL DNAJ HOMOLOG

The DnaK/DnaJ/GrpE heat shock proteins of Escherichia coil constitute the prototype DnaK chaperone machine. Various studies have shown that these three proteins work synergistically in a diverse array of biolog ical functions, including protein folding and disaggregation, proteoly sis, and transport across biological membranes, We have overexpressed and purified the mitochondrial Saccharomyces cerevisiae DnaJ homologue , Mdj1p Delta 55, which lacks the mitochondrial presequence, and studi ed its biochemical properties in well defined in vitro systems, We fin d that Mdj1p Delta 55 interacts with DnaK as judged both by an enzyme- linked immunosorbent assay, as well as stimulation of DnaK's weak ATPa se activity in the presence of GrpE, In addition, Mdj1p Delta 55 not o nly interacts with denatured firefly luciferase on its own, but also e nables DnaK to bind to it in an ATP-dependent mode, Using co-immunopre cipitation assays we can demonstrate the presence of a stable Mdj1p De lta 55-luciferase-DnaK complex. However, in contrast to DnaJ, Mdj1p De lta 55 does not appear to interact well with certain seemingly folded proteins, such as the sigma (32) heat shock transcription factor or th e lambda P DNA replication protein, Finally, Mdj1p Delta 55 can substi tute perfectly well for DnaJ in the refolding of denatured firefly luc iferase by the DnaK chaperone machine. These studies demonstrate that Mdj1p Delta 55 has conserved most of DnacJ's known biological properti es, thus supporting an analogous functional role in yeast mitochondria .