Isolation and characterisation of a cDNA encoding rat mitochondrial GrpE, a stress‐inducible nucleotide‐exchange factor of ubiquitous appearance in mammalian organs

Abstract

In contrast to the E. coli chaperones DnaK, GroEL and GroES, cDNAs encoding mitochondrial homologues of DnaJ and GrpE from higher eukaryotes have yet to be reported. Based on peptide sequences, we have isolated a cDNA encoding a 217 residue nuclear encoded precursor of rat mitochondrial GrpE (mt-GrpE) including a typical mitochondrial presequence of 27 residues. Western blotting revealed that the 21 kDa GrpE homologue is present exclusively in the mitochondrial fraction where it comprises only ~ 0.03% of the total soluble protein, while Northern blotting showed that the mt-GrpE transcript is present in most if not all organs. By contrast to other mitochondrial chaperones, the levels of mt-GrpE and its transcript in cultured cells are only marginally increased in response to the proline analog L-azetidine 2-carboxylic acid but not by heat shock. Furthermore, members of the GrpE family exhibit a much lower degree of sequence identity than do the well studied members of the Hsp70, Hsp60 and Hspl0 families.

The Chloroplastic GrpE Homolog of Chlamydomonas: Two Isoforms Generated by Differential Splicing

“…When compared with 13 additional bacterial GrpE sequences (Naylor et al, 1996), only 5 residues among a total of 32 GrpE sequences were found to be strictly conserved. Furthermore, three residues (Y176, Y180, and R212) in Chlamydomonas CGE1 and one residue (F188) in yeast Mge1p were identified that occur exclusively in cyanobacterial/chloroplastic and mitochondrial GrpE homologs, respectively; therefore, they may provide specific signatures for these homologs (Figure 10, bottom line of alignment).…”

“…In E. coli , the GrpE protein is induced by heat stress (Ang et al, 1986), but this is not the case for its mitochondrial counterparts from yeast or rat (Ikeda et al, 1994;Naylor et al, 1996). To study the expression pattern of the CGE1 gene, we analyzed the accumulation of the CGE1 transcript in Chlamydomonas cells that had been shifted from 25 to 40 Њ C or that had been shifted to the light after 15 hr of dark adaptation.…”

“…Thus, on a molar basis, there is approximately six times more HSP70B than CGE1 present in the stroma. The ratios of Hsp70 to GrpE reported in E. coli and mitochondria are between 3:1 and 9:1 (Neidhardt et al, 1984;Nakai et al, 1994;Voos et al, 1994;Naylor et al, 1996). (C) Reverse transcriptase-mediated PCR analysis of the two CGE1 mRNAs.…”

“…However, the same antiserum failed to cross-react with CGE1 coimmunoprecipitated with HSP70B, and conversely, our CGE1 antiserum did not detect GrpE from total E. coli protein (data not shown). In addition, the seven mature chloroplastic GrpE homologs shown in Figure 10 have calculated molecular masses between 24 and 29 kD, whereas the smallest GrpE protein detected so far, from Lactococcus lactis (Naylor et al, 1996), has a mass of 20.4 kD. Because GrpE proteins generally migrate with apparent molecular masses ‫1ف‬ to 2 kD larger than their actual mass (Zylicz et al, 1987;Bolliger et al, 1994;Deloche and Georgopoulos, 1996;Padidam et al, 1999) (Figures 1, 3, 8, and 9), we consider it unlikely that the 18-kD pea protein that cross-reacted with the E. coli GrpE antiserum was a bona fide GrpE homolog.…”

“…Although functionally similar, Bag-1M and GrpE are not homologous. GrpE homologs have been discovered in mitochondria, first in yeast (Bolliger et al, 1994;Ikeda et al, 1994;Laloraya et al, 1994) and later also in Neurospora (Voos et al, 1994), mammals (Naylor et al, 1996), and plants (Padidam et al, 1999). Because of the presence of a GrpE protein in cyanobacteria, the putative progenitors of today's chloroplasts, and because of the high similarity of chloroplastic Hsp70s with DnaK (Drzymalla et al, 1996), a GrpE homolog also was likely to exist in the chloroplast.…”