Tina Junne

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.

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.

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 precursors basic and amphiphilic targeting signal to acidic receptor domains on both sides of the membrane.

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

ABSTRACT 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.

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° C.

The hydrophobic core of the Sec61 translocon defines the hydrophobicity threshold for membrane integration.

Mutation of the apolar constriction of the yeast Sec61 translocon to polar or charged residues, while retaining functionality, affected the integration of potential transmembrane segments into the lipid bilayer. This indicates that the translocon plays an active role in setting the hydrophobicity threshold for membrane integration.

Mutations in the Sec61p channel affecting signal sequence recognition and membrane protein topology

The orientation of most single-spanning membrane proteins obeys the “positive-inside rule”, i.e. the flanking region of the transmembrane segment that is more positively charged remains in the cytosol. These membrane proteins are integrated by the Sec61/SecY translocon, but how their orientation is achieved is unknown. We have screened for mutations in yeast Sec61p that alter the orientation of single-spanning membrane proteins. We identified a class of mutants that are less efficient in retaining the positively charged flanking region in the cytosol. Surprisingly, these mutations are located at many different sites in the Sec61/SecY molecule, and they do not only involve charged amino acid residues. All these mutants have a prl phenotype that so far have only been seen in bacteria; they allow proteins with defective signal sequences to be translocated, likely because the Sec61p channel opens more easily. A similar correlation between topology defects and prl phenotype was also seen with previously identified yeast Sec61 mutants. Our results suggest a model in which the regulated opening of the translocon is required for the faithful orientation of membrane proteins.

Decatransin, a new natural product inhibiting protein translocation at the Sec61/SecYEG translocon

ABSTRACT A new cyclic decadepsipeptide was isolated from Chaetosphaeria tulasneorum with potent bioactivity on mammalian and yeast cells. Chemogenomic profiling in S. cerevisiae indicated that the Sec61 translocon complex, the machinery for protein translocation and membrane insertion at the endoplasmic reticulum, is the target. The profiles were similar to those of cyclic heptadepsipeptides of a distinct chemotype (including HUN-7293 and cotransin) that had previously been shown to inhibit cotranslational translocation at the mammalian Sec61 translocon. Unbiased, genome-wide mutagenesis followed by full-genome sequencing in both fungal and mammalian cells identified dominant mutations in Sec61p (yeast) or Sec61&agr;1 (mammals) that conferred resistance. Most, but not all, of these mutations affected inhibition by both chemotypes, despite an absence of structural similarity. Biochemical analysis confirmed inhibition of protein translocation into the endoplasmic reticulum of both co- and post-translationally translocated substrates by both chemotypes, demonstrating a mechanism independent of a translating ribosome. Most interestingly, both chemotypes were found to also inhibit SecYEG, the bacterial Sec61 translocon homolog. We suggest ‘decatransin’ as the name for this new decadepsipeptide translocation inhibitor.

Functional asymmetry within the Sec61p translocon

Significance The Sec61/SecY translocon mediates translocation of hydrophilic amino acid sequences across the membrane and integration of hydrophobic segments as transmembrane helices into the lipid bilayer. The integration process is proposed to correspond to thermodynamic equilibration of the translocating sequence between the translocon and the membrane. Here we probed the conditions in the translocon interior in vivo by scanning a cluster of hydrophobic amino acids through the potential transmembrane segment, scoring for membrane insertion vs. translocation. The results reveal functional asymmetry within the translocon caused by the residues forming the central constriction in the translocation pore. Molecular dynamics simulations correlate the insertion behavior with the hydration profile through the pore. The Sec61 translocon forms a pore to translocate polypeptide sequences across the membrane and offers a lateral gate for membrane integration of hydrophobic (H) segments. A central constriction of six apolar residues has been shown to form a seal, but also to determine the hydrophobicity threshold for membrane integration: Mutation of these residues in yeast Sec61p to glycines, serines, aspartates, or lysines lowered the hydrophobicity required for integration; mutation to alanines increased it. Whereas four leucines distributed in an oligo-alanine H segment were sufficient for 50% integration, we now find four leucines in the N-terminal half of the H segment to produce significantly more integration than in the C-terminal half, suggesting functional asymmetry within the translocon. Scanning a cluster of three leucines through an oligo-alanine H segment showed high integration levels, except around the position matching that of the hydrophobic constriction in the pore where integration was strongly reduced. Both asymmetry and the position effect of H-segment integration disappeared upon mutation of the constriction residues to glycines or serines, demonstrating that hydrophobicity at this position within the translocon is responsible for the phenomenon. Asymmetry was largely retained, however, when constriction residues were replaced by alanines. These results reflect on the integration mechanism of transmembrane domains and show that membrane insertion of H segments strongly depends not only on their intrinsic hydrophobicity but also on the local conditions in the translocon interior. Thus, the contribution of hydrophobic residues in the H segment is not simply additive and displays cooperativeness depending on their relative position.

Orientation of Internal Signal-Anchor Sequences at the Sec61 Translocon

Translocation and insertion of secretory and membrane proteins at the endoplasmic reticulum are mediated by the Sec61 translocon. Evidence from in vivo as well as in vitro experiments indicates that N-terminal signal-anchor sequences initially insert N-first before they invert their orientation to translocate the C-terminus. Inversion is driven by flanking charges according to the positive-inside rule and inhibited by increased signal hydrophobicity. Here, we show that upon extending the N-terminal hydrophilic domain preceding the signal core to more than ~20 residues, the insertion behavior changes. Apparent signal inversion and the effect of hydrophobicity are largely lost, suggesting that N-first insertion is limited to N-terminal signal anchors. Extended N-domains sterically hinder N-translocation in a length-dependent manner also for reverse signal anchors with inverted flanking charges. The results indicate a mechanistic difference in the insertion process of N-terminal and internal signal sequences.