Evidence for the Existence of Distinct Mammalian Cytosolic, Microsomal, and Two Mitochondrial GrpE-like Proteins, the Co-chaperones of Specific Hsp70 Members

Abstract

We previously reported the cDNA cloning and characterization of a mammalian mitochondrial GrpE protein (ϳ21 kDa, mt-GrpE#1) and now provide evidence for the presence of distinct cytosolic (ϳ40 kDa), microsomal (ϳ50 kDa), and additional mitochondrial (ϳ22 kDa, mtGrpE#2) GrpE-like members. While a cytosolic GrpElike protein has recently been identified, the demonstration of both a microsomal and a second mitochondrial GrpE-like member represents the first in any biological system. Investigation of the microsomal and two mitochondrial GrpE-like proteins revealed that they bound specifically to Escherichia coli DnaK, and the complexes formed were not disrupted in the presence of 0.5 M salt but were readily dissociated in the presence of 5 mM ATP. The functional integrity of mt-GrpE#1 and #2 was verified by their ability to specifically interact with and stimulate the ATPase activity of mammalian mitochondrial Hsp70 (mt-Hsp70). Analysis of the cDNA sequences encoding the two mammalian mitochondrial GrpE-like proteins revealed ϳ47% positional identity at the amino acid level, the presence of a highly conserved mitochondrial leader sequence, and putative destabilization elements within the 3-untranslated region of the mtGrpE#2 transcript which are not present in the mtGrpE#1 transcript. A constitutive expression of both mitochondrial GrpE-like transcripts in 22 distinct mouse tissues was observed but possible different posttranscriptional regulation of the mt-GrpE#1 and #2 transcripts may confer a different expression pattern of the encoded proteins.

Identification of CHIP, a Novel Tetratricopeptide Repeat-Containing Protein That Interacts with Heat Shock Proteins and Negatively Regulates Chaperone Functions

“…Given the interactions of CHIP with Hsc70 and Hsp70, we examined whether hCHIP had an effect on their ATPase and chaperone activities, in the presence or absence of Hsp40. Recombinant hCHIP expressed in bacteria was used in these studies, given the precedence for retention of ATPase and folding activity when other proteins have been expressed in this system (17,26,27). We confirmed that recombinant hCHIP retained binding activity to Hsc70, to ensure that it is correctly folded and processed in bacteria (not shown).…”

Recent Gene Duplication and Subfunctionalization Produced a Mitochondrial GrpE, the Nucleotide Exchange Factor of the Hsp70 Complex, Specialized in Thermotolerance to Chronic Heat Stress in Arabidopsis

“…Results from this study can provide basic information regarding the evolution of the chaperone machineries. Of note, two copies of Mges were found in rodents and human (Naylor et al, 1998;Oliveira et al, 2006). How and why two Mges evolved in these organisms is unknown.…”

On the brotherhood of the mitochondrial chaperones mortalin and heat shock protein 60

“…The nucleotide exchange factor GrpE enables the recycling of Hsp70 back into an ATP-bound state, permitting the efficient release of its substrate (Har-rison et al 1997). Multiple GrpE-like proteins and a unique human GrpE homolog, HMGE, have been reported to be restricted to the mitochondria and to form chaperone pairing with mortalin/mtHsp70 (Naylor et al 1998;Choglay et al 2001). Besides the known mtDnaJ (Zhao et al 2002), dj2 and dj3 could be candidate cochaperones of mortalin, because these cytosolic DnaJs are also detected in the mitochondria (Terada and Mori 2000).…”