Tim18p, a new subunit of the TIM22 complex that mediates insertion of imported proteins into the yeast mitochondrial inner membrane.

Import of carrier proteins from the cytoplasm into the mitochondrial inner membrane of yeast is mediated by a distinct system consisting of two soluble 70-kDa protein complexes in the intermembrane space and a 300-kDa complex in the inner membrane, the TIM22 complex. The TIM22 complex contains the peripheral subunits Tim9p, Tim10p, and Tim12p and the integral membrane subunits Tim22p and Tim54p. We identify here an additional subunit, an 18-kDa integral membrane protein termed Tim18p. This protein is made as a 21.9-kDa precursor which is imported into mitochondria and processed to its mature form. When mitochondria are gently solubilized, Tim18p comigrates with the other subunits of the TIM22 complex on nondenaturing gels and is coimmunoprecipitated with Tim54p and Tim12p. Tim18p does not cofractionate with the TIM23 complex upon immunoprecipitation or nondenaturing gel electrophoresis. Deletion of Tim18p decreases the growth rate of yeast cells by a factor of two and is synthetically lethal with temperature-sensitive mutations in Tim9p or Tim10p. It also impairs the import of several precursor proteins into isolated mitochondria, and lowers the apparent mass of the TIM22 complex. We suggest that Tim18p functions in the assembly and stabilization of the TIM22 complex but does not directly participate in protein insertion into the inner membrane.

Human deafness dystonia syndrome is a mitochondrial disease.

The human deafness dystonia syndrome results from the mutation of a protein (DDP) of unknown function. We show now that DDP is a mitochondrial protein and similar to five small proteins (Tim8p, Tim9p, Tim10p, Tim12p, and Tim13p) of the yeast mitochondrial intermembrane space. Tim9p, Tim10p, and Tim12p mediate the import of metabolite transporters from the cytoplasm into the mitochondrial inner membrane and interact structurally and functionally with Tim8p and Tim13p. DDP is most similar to Tim8p. Tim8p exists as a soluble 70-kDa complex with Tim13p and Tim9p, and deletion of Tim8p is synthetically lethal with a conditional mutation in Tim10p. The deafness dystonia syndrome thus is a novel type of mitochondrial disease that probably is caused by a defective mitochondrial protein-import system.

Tim9p, an essential partner subunit of Tim10p for the import of mitochondrial carrier proteins.

Tim10p, a protein of the yeast mitochondrial intermembrane space, was shown previously to be essential for the import of multispanning carrier proteins from the cytoplasm into the inner membrane. We now identify Tim9p, another essential component of this import pathway. Most of Tim9p is associated with Tim10p in a soluble 70 kDa complex. Tim9p and Tim10p co-purify in successive chromatographic fractionations and co-immunoprecipitated with each other. Tim9p can be cross-linked to a partly translocated carrier protein. A small fraction of Tim9p is bound to the outer face of the inner membrane in a 300 kDa complex whose other subunits include Tim54p, Tim22p, Tim12p and Tim10p. The sequence of Tim9p is 25% identical to that of Tim10p and Tim12p. A Ser67-->Cys67 mutation in Tim9p suppresses the temperature-sensitive growth defect of tim10-1 and tim12-1 mutants. Tim9p is a new subunit of the TIM machinery that guides hydrophobic inner membrane proteins across the aqueous intermembrane space.

Translocation arrest of an intramitochondrial sorting signal next to Tim11 at the inner-membrane import site.

The import of proteins from the cytosol into the mitochondrial matrix involves the concerted action of two separate import systems: the TOM system in the outer membrane, and the TIM system in the inner membrane. Here we report that the inner-membrane system also sorts proteins to the intermembrane space. Some intermembrane-space proteins, such as cytochromes b2 and c1, are synthesized with a complex pre-sequence consisting of a positively charged matrix targeting signal followed by an uncharged sequence that acts as sorting signal for the intermembrane space. We show that this sorting signal can be efficiently crosslinked to an inner-membrane protein of relative molecular mass 11K after the mature part of the precursor has been sorted to the intermembrane space. The 11K protein, which we term Tim11, is a component of the protein import system in the inner membrane.

Acidic receptor domains on both sides of the outer membrane mediate translocation of precursor proteins into yeast mitochondria.

Mitochondrial precursor proteins made in the cytosol bind to a hetero-oligomeric protein import receptor on the mitochondrial surface and then pass through the translocation channel across the outer membrane. This translocation step is accelerated by an acidic domain of the receptor subunit Mas22p, which protrudes into the intermembrane space. This 'trans' domain of Mas22p specifically binds functional mitochondrial targeting peptides with a Kd of < 1 microM and is required to anchor the N-terminal targeting sequence of a translocation-arrested precursor in the intermembrane space. If this Mas22p domain is deleted, respiration-driven growth of the cells is compromised and import of different precursors into isolated mitochondria is inhibited 3- to 8-fold. Binding of precursors to the mitochondrial surface appears to be mediated by cytosolically exposed acidic domains of the receptor subunits Mas20p and Mas22p. Translocation of a precursor across the outer membrane thus appears to involve sequential binding of the precursor's basic and amphiphilic targeting signal to acidic receptor domains on both sides of the membrane.

Mas37p, a novel receptor subunit for protein import into mitochondria.

By screening a collection of Saccharomyces cerevisiae mutants temperature sensitive for growth on a nonfermentable carbon source, we have isolated a gene (termed MAS37) which encodes a novel receptor for protein import into mitochondria. Mas37p is a 37-kD outer membrane protein with two putative membrane-spanning regions. Inactivation of the MAS37 gene renders cells temperature-sensitive for respiration-driven growth, inhibits import of precursors into isolated mitochondria, and is synthetically lethal with a deletion of one of the genes encoding the import receptors Mas70p or Mas20p. Inactivation of Mas37p with specific antibodies inhibits import of different precursors to different extents; the precursor specificity of Mas37p resembles that of the previously described import receptor Mas70p. Mas70p and Mas37p form a 1:1 complex in detergent extracts of mitochondria and overexpression of one protein enhances that of the other. We suggest that the Mas37p/Mas70p heterodimer functions as a receptor for protein import into yeast mitochondria and that the mitochondrial receptor system consists of hetero-oligomeric subcomplexes with distinct binding activities, but overlapping precursor specificities.

The mitochondrial outer membrane protein Mas22p is essential for protein import and viability of yeast.

We have cloned the gene encoding the protein Mas22p, which spans the outer membrane of yeast mitochondria. Cells that completely lack Mas22p are inviable. The plasmid-borne MAS22 gene suppresses several defects resulting from the deletion of one or more of the mitochondrial protein import receptors. Defects of Mas20p-deficient cells are explained by the reduced level of Mas22p in these mutants. Mas22p has one acidic domain in the cytosol and a second acidic domain in the mitochondrial intermembrane space. We suggest that these domains of Mas22p on either side of the outer membrane function as a relay system for transferring the basic targeting sequences of precursor proteins into the mitochondria.

Yeast mitochondria lacking the two import receptors Mas20p and Mas70p can efficiently and specifically import precursor proteins.

Mas20p and Mas70p are integral proteins of the yeast mitochondrial outer membrane that appear to function as receptors for precursor proteins imported from the cytosol. Loss of either receptor alone does not block import or kill the cells, but deletion of Mas20p causes loss of respiration (Ramage, L., Junne, T., Hahne, K., Lithgow, T., and Schatz, G. (1993) EMBO J. 12, 4115-4123). Here we show that this respiratory deficiency is only temporary; given time to adapt, virtually all cells lacking MAS20p regain respiration without regaining MAS20p. The respiratory defect can also be suppressed (at a frequency of about 10(-6)) by a dominant mutation of a single nuclear gene. The suppressed cells, unlike the unsuppressed ones, tolerate disruption of the MAS70 gene. The resulting double disruptants lack both Mas20p and Mas70p, yet are viable and able to respire. Protein import into mitochondria isolated from these cells is efficient, specific, and highly sensitive to protease treatment. We propose that at least one additional mitochondrial surface protein can function as a protein import receptor and that the activity of this component is up-regulated by a stress response or by an extragenic suppressor.

Cloning and disruption of the gene encoding yeast mitochondrial chaperonin 10, the homolog of E. coli groES.

The mitochondrial chaperonin system consists of chaperonin 60 (also termed hsp60), which is homologous to E. coli groEL, and chaperonin 10, which is homologous to E. coli groES. In yeast, chaperonin 60 function has been shown to be essential for viability. We report here that the same is true for chaperonin 10. We have cloned, sequenced and disrupted the nuclear chaperonin 10 gene CPN10 from Saccharomyces cerevisiae. This gene encodes a protein of 11,372 Da that is imported into the mitochondrial matrix without detectable cleavage. Haploid cells lacking a functional copy of CPN10 fail to grow at temperatures between 23 and 37 degrees C.

Functional cooperation of mitochondrial protein import receptors in yeast.

We have identified a 20 kDa yeast mitochondrial outer membrane protein (termed MAS20) which appears to function as a protein import receptor. We cloned, sequenced and physically mapped the MAS20 gene and found that the protein is homologous to the MOM19 import receptor from Neurospora crassa. MAS20 and MOM19 contain the sequence motif F-X-K-A-L-X-V/L, which is repeated several times with minor variations in the MAS70/MOM72 receptors. To determine how MAS20 functions together with the previously identified yeast receptor MAS70, we constructed yeast mutants lacking either one or both of the receptors. Deletion of either receptor alone had little or no effect on fermentative growth and only partially inhibited mitochondrial protein import in vivo. Deletion of both receptors was lethal. Deleting only MAS70 did not affect respiration; deleting only MAS20 caused loss of respiration, but respiration could be restored by overexpressing MAS70. Import of the F1-ATPase beta-subunit into isolated mitochondria was only partly inhibited by IgGs against either MAS20 or MAS70, but both IgGs inhibited import completely. We conclude that the two receptors have overlapping specificities for mitochondrial precursor proteins and that neither receptor is by itself essential.