Pex19 is involved in importing dually targeted tail-anchored proteins to both mitochondria and peroxisomes.

Tail-anchored (TA) proteins are embedded into their corresponding membrane via a single transmembrane segment at their C-terminus whereas the majority of the protein is facing the cytosol. So far, cellular factors that mediate the integration of such proteins into the mitochondrial outer membrane were not found. Using budding yeast as a model system, we identified the cytosolic Hsp70 chaperone Ssa1 and the peroxisome import factor Pex19 as import mediators for a subset of mitochondrial TA proteins. Accordingly, deletion of PEX19 results in: (1) growth defect under respiration conditions, (2) alteration in mitochondrial morphology, (3) reduced steady-state levels of the mitochondrial TA proteins Fis1 and Gem1, and (4) hampered in organello import of the TA proteins Fis1 and Gem1. Furthermore, recombinant Pex19 can bind directly the TA proteins Fis1 and Gem1. Collectively, this work identified the first factors that are involved in the biogenesis of mitochondrial TA proteins and uncovered an unexpected function of Pex19.

An assay to monitor the membrane integration of single-span proteins.

In vitro import experiments with isolated organelles are a powerful tool for investigation of the biogenesis of proteins. A key issue in such experiments is an assay to distinguish between correctly and incorrectly imported proteins. Here we describe an assay to monitor in vitro the proper membrane integration of single-span proteins. In this assay non-imported proteins are distinguished from correctly imported protein species by labelling of unprotected cysteine residues and a resulting migration shift in SDS-PAGE.

Ergosterol content specifies targeting of tail-anchored proteins to mitochondrial outer membranes.

Tail-anchored (TA) proteins have a single C-terminal transmembrane domain, making their biogenesis dependent on posttranslational translocation. Despite their importance, no dedicated insertion machinery has been uncovered for mitochondrial outer membrane (MOM) TA proteins. To decipher the molecular mechanisms guiding MOM TA protein insertion, we performed two independent systematic microscopic screens in which we visualized the localization of model MOM TA proteins on the background of mutants in all yeast genes. We could find no mutant in which insertion was completely blocked. However, both screens demonstrated that MOM TA proteins were partially localized to the endoplasmic reticulum (ER) in spf1 cells. Spf1, an ER ATPase with unknown function, is the first protein shown to affect MOM TA protein insertion. We found that ER membranes in spf1 cells become similar in their ergosterol content to mitochondrial membranes. Indeed, when we visualized MOM TA protein distribution in yeast strains with reduced ergosterol content, they phenocopied the loss of Spf1. We therefore suggest that the inherent differences in membrane composition between organelle membranes are sufficient to determine membrane integration specificity in a eukaryotic cell.

A crucial role for Mim2 in the biogenesis of mitochondrial outer membrane proteins.

Most of the mitochondrial outer membrane (MOM) proteins contain helical transmembrane domains. Some of the single-span proteins and all known multiple-span proteins are inserted into the membrane in a pathway that depends on the MOM protein Mitochondrial Import 1 (Mim1). So far it has been unknown whether additional proteins are required for this process. Here, we describe the identification and characterization of Mim2, a novel protein of the MOM that has a crucial role in the biogenesis of MOM helical proteins. Mim2 physically and genetically interacts with Mim1, and both proteins form the MIM complex. Cells lacking Mim2 exhibit a severely reduced growth rate and lower steady-state levels of helical MOM proteins. In addition, absence of Mim2 leads to compromised assembly of the translocase of the outer mitochondrial membrane (TOM complex), hampered mitochondrial protein import, and defects in mitochondrial morphology. In summary, the current study demonstrates that Mim2 is a novel central player in the biogenesis of MOM proteins.

Multispan mitochondrial outer membrane protein Ugo1 follows a unique Mim1-dependent import pathway.

The mitochondrial outer membrane (MOM) harbors several multispan proteins that execute various functions. Despite their importance, the mechanisms by which these proteins are recognized and inserted into the outer membrane remain largely unclear. In this paper, we address this issue using yeast mitochondria and the multispan protein Ugo1. Using a specific insertion assay and analysis by native gel electrophoresis, we show that the import receptor Tom70, but not its partner Tom20, is involved in the initial recognition of the Ugo1 precursor. Surprisingly, the import pore formed by the translocase of the outer membrane complex appears not to be required for the insertion process. Conversely, the multifunctional outer membrane protein mitochondrial import 1 (Mim1) plays a central role in mediating the insertion of Ugo1. Collectively, these results suggest that Ugo1 is inserted into the MOM by a novel pathway in which Tom70 and Mim1 contribute to the efficiency and selectivity of the process.

Genetic and functional interactions between the mitochondrial outer membrane proteins Tom6 and Sam37.

The TOM complex is the general mitochondrial entry site for newly synthesized proteins. Precursors of beta-barrel proteins initially follow this common pathway and are then relayed to the SAM/TOB complex, which mediates their integration into the outer membrane. Three proteins, Sam50 (Tob55), Sam35 (Tob38/Tom38), and Sam37 (Mas37), have been identified as the core constituents of the latter complex. Sam37 is essential for growth at elevated temperatures, but the function of the protein is currently unresolved. To identify interacting partners of Sam37 and thus shed light on its function, we screened for multicopy suppressors of sam37Delta. We identified the small subunit of the TOM complex, Tom6, as such a suppressor and found a tight genetic interaction between the two proteins. Overexpression of SAM37 suppresses the growth phenotype of tom6Delta, and cells lacking both genes are not viable. The ability of large amounts of Tom6 to suppress the sam37Delta phenotype can be linked to the capacity of Tom6 to stabilize Tom40, an essential beta-barrel protein which is the central component of the TOM complex. Our results suggest that Sam37 is required for growth at higher temperatures, since it enhances the biogenesis of Tom40, and this requirement can be overruled by improved stability of newly synthesized Tom40 molecules.

Plant mitochondrial rhomboid, AtRBL12, has different substrate specificity from its yeast counterpart.

Rhomboid proteins comprise a class of serine proteases that are conserved in all kingdoms of organisms. They contain six or seven transmembrane helices and control a wide range of cellular functions and developmental processes by intramembrane proteolysis. This paper provides experimental evidence for the existence of rhomboid proteases in plant mitochondria and chloroplasts. Among 15 putative rhomboid-like proteins in Arabidopsis thaliana, we selected five predicted as mitochondrially targeted. For these proteins we performed the GFP transient assay, and identified two homologues, AtRBL11 (At5g25752) and AtRBL12 (At1g18600) to be targeted into plastids and mitochondria, respectively. Phylogenetic analysis reveals that AtRBL12 or AtRBL11 have only one clear orthologue in plant species with completely sequenced genomes. Complementation of the yeast lacking a functional copy of mitochondrial rhomboid with AtRBL12 indicates that this plant protease, in contrast to the human orthologue, does not recognize the yeast substrates, cytochrome c peroxidase (Ccp1) or dynamin-like GTPase (Mgm1). In agreement with this, we did not observe processing of Mgm1 when labeled precursor of this protein was incubated in vitro with Arabidopsis mitochondrial extract. Our results imply that plant mitochondrial rhomboids function in a specific manner and thus differ from their yeast and mammal counterparts.