Purification and biochemical properties of Saccharomyces cerevisiae Mdj1p, the mitochondrial DnaJ homologue.
The DnaK/DnaJ/GrpE heat shock proteins of Escherichia coli constitute the prototype DnaK chaperone machine. Various studies have shown that these three proteins work synergistically in a diverse array of biological functions, including protein folding and disaggregation, proteolysis, and transport across biological membranes. We have overexpressed and purified the mitochondrial Saccharomyces cerevisiae DnaJ homologue, Mdj1pDelta55, which lacks the mitochondrial presequence, and studied its biochemical properties in well defined in vitro systems. We find that Mdj1pDelta55 interacts with DnaK as judged both by an enzyme-linked immunosorbent assay, as well as stimulation of DnaK's weak ATPase activity in the presence of GrpE. In addition, Mdj1pDelta55 not only interacts with denatured firefly luciferase on its own, but also enables DnaK to bind to it in an ATP-dependent mode. Using co-immunoprecipitation assays we can demonstrate the presence of a stable Mdj1pDelta55-luciferase-DnaK complex. However, in contrast to DnaJ, Mdj1pDelta55 does not appear to interact well with certain seemingly folded proteins, such as the sigma32 heat shock transcription factor or the lambdaP DNA replication protein. Finally, Mdj1pDelta55 can substitute perfectly well for DnaJ in the refolding of denatured firefly luciferase by the DnaK chaperone machine. These studies demonstrate that Mdj1pDelta55 has conserved most of DnaJ's known biological properties, thus supporting an analogous functional role in yeast mitochondria.
Deloche
O
O
Département de Biochimie Médicale, Centre Médical Universitaire, 1, rue Michel-Servet, 1211 Geneva 4, Switzerland. olivier.deloche@medecine.unige.ch
Liberek
K
K
Zylicz
M
M
Georgopoulos
C
C
D000251:Adenosine Triphosphatases / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D029968:Escherichia coli Proteins; D005656:Fungal Proteins / Q000302:isolation & purification*; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins
C494635:DnaJ protein, E coli; D029968:Escherichia coli Proteins; D005656:Fungal Proteins; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; C087041:MDJ1 protein, S cerevisiae; D008565:Membrane Proteins; D029701:Saccharomyces cerevisiae Proteins; D008156:Luciferases; D000251:Adenosine Triphosphatases; C063842:dnaK protein, E coli