The outer envelope protein OEP24 from pea chloroplasts can functionally replace the mitochondrial VDAC in yeast

The chloroplastic outer envelope protein OEP24 from pea forms a high-conductance low specificity solute channel as shown by in vitro studies. In order to establish its function also in an in vivo-like system, the gene encoding OEP24 was transformed into a yeast strain which lacks the general mitochondria solute channel porin, also known as voltage-dependent anion channel (VDAC). Transformation of the yeast VDAC(-) strain with the OEP24 gene resulted in the recovery of a phenotype indistinguishable from the wild-type. The OEP24 polypeptide is targeted to the mitochondrial outer membrane in this heterologous system. We conclude that OEP24 forms a solute channel in pea chloroplasts in planta.     

A mitochondrial presequence can transport a chloroplast-encoded protein into yeast mitochondria

Ribulose-1,5-bisphosphate carboxylase/oxygenase of chloroplasts contains eight large and eight small subunits. The small subunit is encoded by nuclear DNA, synthesized in the cytoplasm, and imported into chloroplasts. The large subunit is encoded by chloroplast DNA and synthesized within chloroplasts. We show in this communication that the large subunit of Chlamydomonas chloroplasts could be efficiently imported into isolated yeast mitochondria if it was attached to the presequence of a protein transported into the yeast mitochondrial matrix. Thus, synthesis of the large subunit within chloroplasts does not reflect the inability of this subunit to cross membranes. The same mitochondrial presequence could also transport the nuclear-encoded small subunit into yeast mitochondria. However, when the two types of subunits were coimported into mitochondria, they did not assemble with each other inside the heterologous organelle.     

A yeast mitochondrial leader peptide functions in vivo as a dual targeting signal for both chloroplasts and mitochondria.

A fusion protein was expressed in transgenic tobacco and yeast cells to examine the functional conservation of mechanisms for importing precursor proteins from the cytosol into mitochondria and chloroplasts. The test protein consisted of the mitochondrial leader peptide from the yeast precursor to cytochrome oxidase subunit Va (prC5) fused to the reporter protein chloramphenicol acetyltransferase. This protein, denoted prC5/CAT, was transported into the mitochondrial interior in yeast and tobacco cells. In both organisms, the mitochondrial form of prC5/CAT was smaller than the primary translation product, suggesting that proteolytic processing occurred during the transport process. prC5/CAT also was translocated into chloroplasts in vivo, accumulating to approximately the same levels as in plant mitochondria. However, accumulation of prC5/CAT in chloroplasts relative to mitochondria varied with the conditions under which plants were grown. The chloroplast form of prC5/CAT also appeared to have been proteolytically processed, yielding a mature protein of the same apparent size as that seen in mitochondria of either tobacco or yeast. Chloramphenicol acetyltransferase lacking a mitochondrial targeting peptide did not associate with either chloroplasts or mitochondria. The results demonstrated that in plant cells a single leader peptide can interact functionally with the protein translocation systems of both chloroplasts and mitochondria, and raised the possibility that certain native proteins might be shared between these two organelles.     

A novel small-subunit ribosomal protein of yeast mitochondria that interacts functionally with an mRNA-specific translational activator.

Mitochondrial translation of the mRNA encoding cytochrome c oxidase subunit III (coxIII) specifically requires the action of three position activator proteins encoded in the nucleus of Saccharomyces cerevisiae. Some mutations affecting one of these activators, PET122, can be suppressed by mutations in an unlinked nuclear gene termed PET123. PET123 function was previously demonstrated to be required for translation of all mitochondrial gene products. We have now generated an antibody against the PET123 protein and have used it to demonstrate that PET123 is a mitochondrial ribosomal protein of the small subunit. PET123 appears to be present at levels comparable to those of other mitochondrial ribosomal proteins, and its accumulation is dependent on the presence of the 15S rRNA gene in mitochondria. Taken together with the previous genetic data, these results strongly support a model in which the mRNA-specific translational activator PET122 works by directly interacting with the small ribosomal subunit to promote translation initiation on the coxIII mRNA.     

Spi-B can functionally replace PU.1 in myeloid but not lymphoid development

Mature macrophages, neutrophils and lymphoid cells do not develop in PU.1(-/-) mice. In contrast, mice lacking the highly related protein Spi-B generate all hematopoietic lineages but display a B-cell receptor signaling defect. These distinct phenotypes could result from functional differences between PU.1 and Spi-B or their unique temporal and tissue-specific expression (PU.1: myeloid and B cells; Spi-B: B cells only). To address this question, we introduced the Spi-B cDNA into the murine PU.1 locus by homologous recombination. In the absence of PU.1, Spi-B rescued macrophage and granulocyte development when assayed by in vitro differentiation of embryonic stem cells. Adherent, CD11b(+)/F4/80(+) cells capable of phagocytosis were detected in PU.1(Spi-B/Spi-B) embryoid bodies, and myeloid colonies were present in hematopoietic progenitor assays. Despite its ability to rescue myeloid differentiation, Spi-B did not rescue lymphoid development in a RAG-2(-/-) complementation assay. These results demonstrate an important difference between PU.1 and Spi-B. Careful comparison of these Ets factors will delineate important functional domains of PU.1 involved in lymphocyte lineage commitment and/or maturation.     

Coupling of protein synthesis and mitochondrial import in a homologous yeast in vitro system.

We made use of a homologous cell-free mitochondrial protein import system derived from the yeast Saccharomyces cerevisiae to investigate the coupling of protein synthesis and import. Mitochondrial precursor proteins were synthesized in a yeast lysate either in the presence or absence of isolated yeast mitochondria. We were, therefore, able to analyze protein import into mitochondria either in a strictly posttranslational reaction (when isolated mitochondria were added only after protein synthesis has been arrested by the addition of cycloheximide) or in a reaction in which synthesis and import were permitted to occur simultaneously. We found that the import of a precursor protein consisting of the amino-terminal mitochondrial targeting sequence of cytochrome oxidase subunit IV fused to mouse dihydrofolate reductase is very inefficient in a strictly posttranslational reaction, whereas efficient import is observed if precursor synthesis and import are coupled. The same result was obtained when we analyzed the import of bulk endogenous yeast mitochondrial proteins in this system. Finally, we found that the insertion of the yeast outer membrane protein porin is also several times more efficient when synthesis and insertion are coupled.     

In vitro and in vivo studies on the mitochondrial import of CBS1, a translational activator of cytochrome b in yeast

Summary Translation of mitochondrial cytochrome b mRNA in yeast is activated by the product of the nuclear gene CBS1. CBS1 encodes a 27 kDa precursor protein, which is cleaved to a 24 kDa mature protein during the import into isolated mitochondria. The sequences required for mitochondrial import reside in the amino-terminal end of the CBS1 precursor. Deletion of the 76 amino-terminal amino acids renders the protein incompetent for mitochondrial import in vitro and non-functional in vivo. When present on a high copy number plasmid and under the control of a strong yeast promoter, biological function can be restored by this truncated derivative. This observation indicates that the CBS1 protein devoid of mitochondrial targeting sequences can enter mitochondria in vivo, possibly due to a bypass of the mitochondrial import system.     

A yeast mitochondrial membrane methyltransferase-like protein can compensate for oxa1 mutations

Members of the Oxa1p/Alb3/YidC family mediate the insertion of various organelle or bacterial hydrophobic proteins into membranes. They present at least five transmembrane segments (TM) linked by hydrophilic domains located on both sides of the membrane. To examine how Oxa1p structure relates to its function, we have introduced point mutations and large deletions into various domains of the yeast mitochondrial protein. These mutants allowed us to show the importance of the first TM domain as well as a synergistic interaction between the first loop and the C-terminal tail, which both protrude into the matrix. These mutants also led to the isolation of a high copy suppressor, OMS1, which encodes a member of the methyltransferase family. Overexpression of OMS1 seems to increase the steady-state level of both the mutant and wild-type Oxa1p. We show that Oms1p is a mitochondrial inner membrane protein inserted independently of Oxa1p. Oms1p presents one TM and a N-in C-out topology with the C-terminal domain carrying the methyltransferase-like domain. A conserved motif within this domain is essential for the suppression of oxa1 mutations. We discuss the possible role of Oms1p on Oxa1p intermembrane space domain.