Martin Horst

A mitochondrial homolog of bacterial GrpE interacts with mitochondrial hsp70 and is essential for viability.

Mitochondrial hsp70 (mhsp70) is located in the matrix and an essential component of the mitochondrial protein import system. To study the function of mhsp70 and to identify possible partner proteins we constructed a yeast strain in which all mhsp70 molecules carry a C‐terminal hexa‐histidine tag. The tagged mhsp70 appears to be functional in vivo. When an ATP depleted mitochondrial extract was incubated with a nickel‐derivatized affinity resin, the resin bound not only mhsp70, but also a 23 kDa protein. This protein was dissociated from mhsp70 by ATP. ADP and GTP were much less effective in promoting dissociation whereas CTP and TTP were inactive. We cloned the gene encoding the 23 kDa protein. This gene, termed GRPE, encodes a 228 residue protein, whose sequence closely resembles that of the bacterial GrpE protein. Microsequencing the purified 23 kDa protein established it as the product of the yeast GRPE gene. Yeast GrpEp is made as a precursor that is cleaved upon import into isolated mitochondria. GrpEp is essential for viability. We suggest that this protein interacts with mhsp70 in a manner analogous to that of GrpE with DnaK of E.coli.

Sequential action of two hsp70 complexes during protein import into mitochondria

The mitochondrial chaperone mhsp70 mediates protein transport across the inner membrane and protein folding in the matrix. These two reactions are effected by two different mhsp70 complexes. The ADP conformation of mhsp70 favors formation of a complex on the inner membrane; this ‘import complex’ contains mhsp70, its membrane anchor Tim44 and the nucleotide exchange factor mGrpE. The ATP conformation of mhsp70 favors formation of a complex in the matrix; this ‘folding complex’ contains mhsp70, the mitochondrial DnaJ homolog Mdj1 and mGrpE. A precursor protein entering the matrix interacts first with the import complex and then with the folding complex. A chaperone can thus function as part of two different complexes within the same organelle.

Dynamic interaction of the protein translocation systems in the inner and outer membranes of yeast mitochondria.

Mitochondria contain two distinct protein import systems, one in the outer and the other in the inner membrane. These systems can act independently of one another in submitochondrial fractions of if a protein is transported to the outer membrane or to the intermembrane space. It has been proposed that the two systems associate reversibly when a protein is transported across both membranes, but this hypothesis has remained unproven. In order to address this question, we have checked whether antibodies against a subunit of one system can co‐immunoprecipitate subunits of the other system. We find that the two systems associate stably if a matrix‐targeted precursor is arrested during import; no association is seen in the absence of a stuck precursor. These experiments provide direct evidence that protein import into the mitochondrial matrix is mediated by the reversible interaction of the two translocation systems.

Protein import into yeast mitochondria: The inner membrane import site protein ISP45 is the MPI1 gene product

Protein import across both mitochondrial membranes is mediated by the cooperation of two distinct protein transport systems, one in the outer and the other in the inner membrane. Previously we described a 45 kDa yeast mitochondrial inner membrane protein (ISP45) that can be cross‐linked to a partially translocated precursor protein (Scherer et al., 1992). We have now purified ISP45 to homogeneity and identified it as the product of the nuclear MPI1 gene. Identity of ISP45 with the MPI1 gene product was shown by microsequencing of three tryptic ISP45 peptides and by demonstrating that an antibody against an Mpi1p‐beta‐galactosidase fusion protein specifically recognizes ISP45. Antibodies monospecific for ISP45 inhibited protein import into right‐side‐out mitochondrial inner membrane vesicles, but not into intact mitochondria. On solubilizing mitochondria, ISP45 was rapidly converted to a 40 kDa proteolytic fragment unless mitochondria were first denatured with trichloroacetic acid. The combined genetic and biochemical evidence identifies ISP45/Mpi1p as a component of the protein import system of the yeast mitochondrial inner membrane.

The Mas20p and Mas70p subunits of the protein import receptor of yeast mitochondria interact via the tetratricopeptide repeat motif in Mas20p: evidence for a single hetero-oligomeric receptor.

Protein import into yeast mitochondria is mediated by four integral outer membrane proteins which function as import receptors. These proteins (termed Mas20p, Mas22p, Mas37p and Mas70p) appear to exist as two subcomplexes: a Mas37p‐Mas70p heterodimer and a less well characterized Mas20p‐Mas22p complex. The subcomplexes interact functionally during protein import, but it has remained uncertain whether they are in direct contact with each other in vivo. Here we show that Mas20p and Mas70p can be cross‐linked in intact mitochondria, or co‐immunoprecipitated from digitonin‐solubilized mitochondria. Furthermore, the cytosolic domains of these two proteins interact in the ‘two‐hybrid’ system. Association of Mas20p and Mas70p is virtually abolished by a mutation in the single tetratricopeptide motif in Mas20p. This mutation specifically inhibits import of precursors that are first recognized by Mas37p‐Mas70p and only then transferred to Mas20p‐Mas22p. We conclude that the two receptor subcomplexes of the mitochondrial protein import receptor interact in vivo via their Mas20p and Mas70p subunits and that this interaction is functionally important.

WHAT IS THE DRIVING FORCE FOR PROTEIN IMPORT INTO MITOCHONDRIA

Nuclear-encoded mitochondrial proteins are synthesized in the cytosol as precursors and then imported into mitochondria. Protein import into the matrix space requires the function of the mitochondrial hsp70 (mhsp70) chaperone. mhsp70 is an ATPase that acts in conjunction with two partner proteins: the Tim44 subunit of the inner membrane import complex, and the nucleotide exchange factor mGrpE. A central question concerns how mhsp70 uses the energy of ATP hydrolysis to transport precursor proteins into the matrix. Recent evidence suggests that mhsp70 is a mechanochemical enzyme that actively pulls precursors across the inner membrane.

Unraveling the Protein Translocation Machinery in the Mitochondrial Inner Membrane

Publisher Summary This chapter discusses the protein translocation machinery in the mitochondrial inner membrane. Electron microscopy studies revealed the presence of “contact sites” between the outer and inner mitochondrial membranes. There is now general agreement that these “contact sites” are the ports through which cytoplasmically-made precursor proteins enter the mitochondrial matrix. It is reasonable to speculate that a stable channel through, which proteins enter the matrix spans both membranes at contact sites. Dynamic channel model and its implications for protein sorting to the intermembrane space are presented. There are two dynamically interacting import channels. The fimctional independence of the two channels is essential for the import and sorting of several proteins to the intermembrane space. These proteins include CCHL, cytochrome c 1 , cytochrome b 2 , and probably cytochrome c peroxidase and mitochondrial creatine kinase. In addition, the inner mitochondria1 membrane translocation machinery is explored.

Protein import into mitochondria

Abstract Most mitochondrial proteins are encoded by the nucleus and synthesized in the cytoplasm. Protein sorting within mitochondria depends on the precise coordination of several components of the import machinery. In this review we will discuss how precursor proteins are targeted to mitochondria, the energetics of import, intramito-chondrial sorting, the requirement of cytosolic and matrix localized chaperones, as well as the import channels themselves.