Dean J. Naylor

The Role of Molecular Chaperones in Mitochondrial Protein Import and Folding

Molecular chaperones play a critical role in many cellular processes. This review concentrates on their role in targeting of proteins to the mitochondria and the subsequent folding of the imported protein. It also reviews the role of molecular chaperons in protein degradation, a process that not only regulates the turnover of proteins but also eliminates proteins that have folded incorrectly or have aggregated as a result of cell stress. Finally, the role of molecular chaperones, in particular to mitochondrial chaperonins, in disease is reviewed. In support of the endosymbiont theory on the origin of mitochondria, the chaperones of the mitochondrial compartment show a high degree of similarity to bacterial molecular chaperones. Thus, studies of protein folding in bacteria such as Escherichia coli have proved to be instructive in understanding the process in the eukaryotic cell. As in bacteria, the molecular chaperone genes of eukaryotes are activated by a variety of stresses. The regulation of stress genes involved in mitochondrial chaperone function is reviewed and major unsolved questions regarding the regulation, function, and involvement in disease of the molecular chaperones are identified.

Role of chaperones in the biogenesis and maintenance of the mitochondrion.

All cells depend on correctly folded proteins for optimal function. A central question in cellular biology is how such folded structures are formed and maintained, a process that is now recognized to rely heavily on a group of proteins called molecular chaperones. Molecular chaperones constitute distinct families of proteins that are ubiquitous and highly conserved from bacteria to humans. They appear to bind nonnative conformations of most, if not all, proteins, thereby preventing their aggregation and subsequent inactivation. The chaperones not only protect newly synthesized proteins during transport and folding, but also serve to maintain the cell in a healthy state during exposure to a multitude of stress conditions. Accordingly, chaperones are expressed constitutively, but their synthesis is further enhanced during stress conditions. Detailed insights into the role of molecular chaperones have come from studies of mitochondrial protein biogenesis, a process in which chaperones act as unfoldases, pulling devices, and foldases. In this review we summarize these developments and further discuss the potential role of chaperones in mitochondrial DNA metabolism and human mitochondrial disease states.—Martinus, R. D., Ryan, M. T., Naylor, D. J., Herd, S. M., Hoogenraad, N. J., Høj, P. B. Role of chaperones in the biogenesis and maintenance of the mitochondrion. FASEB J. 9, 371–378 (1995)

Evidence for the existence of distinct mammalian cytosolic, microsomal, and two mitochondrial GrpE-like proteins, the co-chaperones of specific Hsp70 members

We previously reported the cDNA cloning and characterization of a mammalian mitochondrial GrpE protein (∼21 kDa, mt-GrpE#1) and now provide evidence for the presence of distinct cytosolic (∼40 kDa), microsomal (∼50 kDa), and additional mitochondrial (∼22 kDa, mt-GrpE#2) GrpE-like members. While a cytosolic GrpE-like 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 0.5m 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 ∼47% 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 mt-GrpE#2 transcript which are not present in the mt-GrpE#1 transcript. A constitutive expression of both mitochondrial GrpE-like transcripts in 22 distinct mouse tissues was observed but possible different post-transcriptional regulation of the mt-GrpE#1 and #2 transcripts may confer a different expression pattern of the encoded proteins.

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

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 Hsp10 families.

Affinity-purification and identification of GrpE homologues from mammalian mitochondria

We used affinity chromatography on DnaK columns to identify a mitochondrial GrpE homologue from bovine, porcine and rat liver mitochondria. The 24 kDa GrpE homologue bound specifically to the DnaK column and was not eluted with 1 M KCl but readily with 5 mM ATP. Sequence analysis of the bovine homologue (85 residues) revealed 42% positional identity to mitochondrial GrpEp from S. cerevisiae and about 30% identity to the bacterial counterparts. Thus, GrpE homologues from higher and lower eukaryotes are highly conserved.

Characterisation of several Hsp70 interacting proteins from mammalian organelles

Since both the spectrum and characteristics of in vivo substrates with affinity for Hsp70 members are largely unknown, we have investigated the range and type of mammalian organellar proteins which selectively interact with immobilised Escherichia coli Hsp70 (DnaK). Amongst a subset of organellar proteins selectively retained on DnaK, the major constituents represent unstable proteins and subunits of oligomeric proteins. The interactions with DnaK were diminished in the presence of mt-Hsp70 and BiP, while the complexes formed with DnaK were dissociated in the presence of K+ and GrpE-like co-chaperones, suggesting that these organellar proteins constitute general Hsp70 substrates. Protein sequence analysis identified the major DnaK interacting constituents as the mitochondrial transcription factor A, the alpha- (but not the beta-) subunit of succinyl CoA synthetase, mitochondrial 2,4-dienoyl CoA reductase, endoplasmic reticulum cyclophilin-B, peroxisomal multifunctional enzyme and a previously undescribed peroxisomal protein suspected to represent an isoform of 2,4-dienoyl CoA reductase. The selective retention of these fully synthesised proteins on Hsp70 most likely reflects the function of this molecular chaperone in protein biogenesis, but additionally, could extend the known functions of Hsp70 to include modulating the activities of certain proteins or enzymes which are important in cellular homeostasis.

Characterization of Novel Hsp70 in Mammalian-Cells

A new protein detected in heat resistant mutants of a murine fibrosarcoma has been identified as a member of the hsp70 family. The protein is similar to the constitutive hsp70 of the parent cells with regard to its antibody cross-reactivity, its ability to bind to an ATP affinity column and partial amino acid sequencing. It is present in addition to, and in similar amounts to, the constitutive isoform of the heat resistant mutant cells and the parent cell line. The lack of either of two post-translational modifications common to other hsps, phosphorylation and ADP-ribosylation, and the demonstration of mRNA for the novel protein suggest that it is a separate gene product and not a post-translational modification of the constitutive hsp70. To our knowledge, this protein has not been described in other systems and may be important in the expression of the heat resistant phenotype of these cells. The in vivo phosphorylation patterns also show that hsp90 and hsp28 are heavily phosphorylated in the heat resistant mutant, but that two other stress proteins reported to be phosphorylated under some conditions are not phosphorylated in these cells.