Accumulation of mitochondrially synthesized Saccharomyces cerevisiae Cox2p and Cox3p depends on targeting information in untranslated portions of their mRNAs.

The essential products of the yeast mitochondrial translation system are seven hydrophobic membrane proteins and Var1p, a hydrophilic protein in the small ribosomal subunit. Translation of the membrane proteins depends on nuclearly encoded, mRNA-specific translational activators that recognize the 5'-untranslated leaders of their target mRNAs. These translational activators are themselves membrane associated and could therefore tether translation to the inner membrane. In this study, we tested whether chimeric mRNAs with the untranslated sequences normally present on the mRNA encoding soluble Var1p, can direct functional expression of coding sequences specifying the integral membrane proteins Cox2p and Cox3p. DNA sequences specifying these chimeric mRNAs were inserted into mtDNA at the VAR1 locus and expressed in strains containing a nuclearly localized plasmid that supplies a functional form of Var1p, imported from the cytoplasm. Although cells expressing these chimeric mRNAs actively synthesized both membrane proteins, they were severely deficient in cytochrome c oxidase activity and in the accumulation of Cox2p and Cox3p, respectively. These data strongly support the physiological importance of interactions between membrane-bound mRNA-specific translational activators and the native 5'-untranslated leaders of the COX2 and COX3 mRNAs for localizing productive synthesis of Cox2p and Cox3p to the inner membrane.     

Release of adenylate kinase 2 from the mitochondrial intermembrane space during apoptosis

The release of two mitochondrial proteins, cytochrome c and apoptosis-inducing factor (AIF), into the soluble cytoplasm of cells undergoing apoptosis is well established. Using spectrophotometric determination of enzyme activity, the accumulation of adenylate kinase (AK) activity in the cytosolic fraction of apoptotic cells has also been observed recently. However, three isozymes, AK1, AK2 and AK3, have been characterized in mammalian cells and shown to be localized in the cytosol, mitochondrial intermembrane space and mitochondrial matrix, respectively, and it is unknown which one of these isozymes accumulates in the cytosol during apoptosis. We now demonstrate that in apoptotic cells only AK2 was translocated into the cytosol concomitantly with cytochrome c. The amount of AK1 in cytosol, as well as the amount of matrix-associated AK3, remained unchanged during the apoptotic process. Thus, our data suggest that only intermembrane proteins are released from mitochondria during the early phase of the apoptotic process.     

The redox environment in the mitochondrial intermembrane space is maintained separately from the cytosol and matrix

Redox control in the mitochondrion is essential for the proper functioning of this organelle. Disruption of mitochondrial redox processes contributes to a host of human disorders, including cancer, neurodegenerative diseases, and aging. To better characterize redox control pathways in this organelle, we have targeted a green fluorescent protein-based redox sensor to the intermembrane space (IMS) and matrix of yeast mitochondria. This approach allows us to separately monitor the redox state of the matrix and the IMS, providing a more detailed picture of redox processes in these two compartments. To verify that the sensors respond to localized glutathione (GSH) redox changes, we have genetically manipulated the subcellular redox state using oxidized GSH (GSSG) reductase localization mutants. These studies indicate that redox control in the cytosol and matrix are maintained separately by cytosolic and mitochondrial isoforms of GSSG reductase. Our studies also demonstrate that the mitochondrial IMS is considerably more oxidizing than the cytosol and mitochondrial matrix and is not directly influenced by endogenous GSSG reductase activity. These redox measurements are used to predict the oxidation state of thiol-containing proteins that are imported into the IMS.     

Export of mitochondrially synthesized lysophosphatidic acid

We have previously demonstrated that the properties of mitochondrial glycerophosphate acyltransferase are in keeping with the asymmetric distribution of fatty acids found in naturally occurring cell glycerophospholipids. We are now examining if mitochondria can export lysophosphatidic acid and if it is converted to other phospholipids by the microsomes. Rat liver mitochondria were incubated for 3 min with [2-3H]-sn-glycerol 3-phosphate, palmityl-CoA, and N-ethylmaleimide in the acyltransferase assay medium. In the absence of bovine serum albumin in the medium, greater than 80% of the phospholipids sedimented with the mitochondria. In the presence of the albumin, the lysophosphatidic acid was present entirely in the supernatant fluid. The very little phosphatidic acid that was formed sedimented with the mitochondria. Addition of microsomes to the supernatant fluid followed by a further incubation of 5 min converted 61% of the lysophosphatidic acid to phosphatidic acid which sedimented with the microsomes. When mitochondria and microsomes were incubated together in the assay medium containing albumin and N-ethylmaleimide, the product contained more phosphatidic and less lysophosphatidic acid. When the subcellular components were reisolated by differential centrifugation, 70% of the phosphatidic acid sedimented with the microsomes and the lysophosphatidic acid stayed in the postmicrosomal supernatant. Thus, under appropriate conditions mitochondrially produced lysophosphatidic acid can leave the organelles and this phospholipid can be converted to phosphatidic acid by the microsomes.     

Redox-regulated dynamic interplay between Cox19 and the copper-binding protein Cox11 in the intermembrane space of mitochondria facilitates biogenesis of cytochrome c oxidase

Members of the twin Cx₉C protein family constitute the largest group of proteins in the intermembrane space (IMS) of mitochondria. Despite their conserved nature and their essential role in the biogenesis of the respiratory chain, the molecular function of twin Cx₉C proteins is largely unknown. We performed a SILAC-based quantitative proteomic analysis to identify interaction partners of the conserved twin Cx₉C protein Cox19. We found that Cox19 interacts in a dynamic manner with Cox11, a copper transfer protein that facilitates metalation of the Cu(B) center of subunit 1 of cytochrome c oxidase. The interaction with Cox11 is critical for the stable accumulation of Cox19 in mitochondria. Cox19 consists of a helical hairpin structure that forms a hydrophobic surface characterized by two highly conserved tyrosine-leucine dipeptides. These residues are essential for Cox19 function and its specific binding to a cysteine-containing sequence in Cox11. Our observations suggest that an oxidative modification of this cysteine residue of Cox11 stimulates Cox19 binding, pointing to a redox-regulated interplay of Cox19 and Cox11 that is critical for copper transfer in the IMS and thus for biogenesis of cytochrome c oxidase.     

Chaperoning through the mitochondrial intermembrane space

The first high-resolution structure of a mitochondrial translocase complex, the Tim9-Tim10 chaperone, is reported by Webb et al. (2006) in a recent issue of Molecular Cell, providing important insight in the transport of hydrophobic proteins through the aqueous intermembrane space and the mechanisms of protein assembly.     

Import of cytochrome b2 to the mitochondrial intermembrane space: the tightly folded heme-binding domain makes import dependent upon matrix ATP.

Cytochrome b2 is synthesized as a precursor in the cytoplasm and imported to the intermembrane space of yeast mitochondria. We show here that the precursor contains a tightly folded heme-binding domain and that translocation of this domain across the outer membrane requires ATP. Surprisingly, it is ATP in the mitochondrial matrix rather than external ATP that drives import of the heme-binding domain. When the folded structure of the heme-binding domain is disrupted by mutation or by urea denaturation, import and correct processing take place in ATP-depleted mitochondria. These results indicate that (1) cytochrome b2 reaches the intermembrane space without completely crossing the inner membrane, and (2) some precursors fold outside the mitochondria but remain translocation-competent, and import of these precursors in vitro does not require ATP-dependent cytosolic chaperone proteins.     

Antifolding activity of hsp60 couples protein import into the mitochondrial matrix with export to the intermembrane space.

Cytochrome b2 reaches the intermembrane space of mitochondria by transport into the matrix followed by export across the inner membrane. While in the matrix, the protein interacts with hsp60, which arrests its folding prior to export. The bacterial-type export sequence in pre-cytochrome b2 functions by inhibiting the ATP-dependent release of the protein from hsp60. Release for export apparently requires, in addition to ATP, the interaction of the signal sequence with a component of the export machinery in the inner membrane. Export can occur before import is complete provided that a critical length of the polypeptide chain has been translocated into the matrix. Thus, hsp60 combines two activities: catalysis of folding of proteins destined for the matrix, and maintaining proteins in an unfolded state to facilitate their channeling between the machineries for import and export across the inner membrane. Anti-folding signals such as the hydrophobic export sequence in cytochrome b2 may act as switches between these two activities.     

Protein transport in chloroplasts - targeting to the intermembrane space

The import of proteins destined for the intermembrane space of chloroplasts has not been investigated in detail up to now. By investigating energy requirements and time courses, as well as performing competition experiments, we show that the two intermembrane space components Tic22 and MGD1 (E.C. 2.4.1.46) both engage the Toc machinery for crossing the outer envelope, whereas their pathways diverge thereafter. Although MGD1 appears to at least partly cross the inner envelope, Tic22 very likely reaches its mature form in the intermembrane space without involving stromal components. Thus, different pathways for intermembrane space targeting probably exist in chloroplasts.     

Shuttle mission in the mitochondrial intermembrane space

Lipid trafficking is essential for biogenesis and maintenance of eukaryotic organelles. In this issue of The EMBO Journal, Saita et al ( 2018 ) revealed that proteolytic processing by the rhomboid protease PARL in the mitochondrial inner membrane facilitates partitioning of START domain‐containing protein STARD7 to the cytosol and mitochondrial intermembrane space. STARD7 in the mitochondrial intermembrane space functions as a lipid transfer protein to shuttle phosphatidylcholine from the outer membrane to the inner membrane.     

Transport of proteins to the mitochondrial intermembrane space: the ''matrix-targeting'' and the ''sorting'' domains in the cytochrome c1 presequence.

We reported earlier that the yeast cytochrome c1 presequence (length: 61 amino acids) directs attached proteins to the mitochondrial intermembrane space and that it appears to contain two functional domains: a 'matrix-targeting' domain, and a 'sorting' domain. We have now used gene manipulation together with two different in vivo import assays to map these two domains within the cytochrome c1 presequence. The 'matrix-targeting' domain is contained within the N-terminal 16 residues (or less); by itself, it directs attached proteins to the matrix. The 'sorting' domain extends into the C-terminal 13 residues of the presequence; while it does not mediate intracellular protein transport by itself, it acts together with the preceding 'matrix-targeting' sequence in sorting attached proteins into the intermembrane space. On replacing the authentic 'matrix-targeting' sequence with artificial sequences of different lengths we found that sorting of proteins between the outer membrane and the intermembrane space is not exclusively determined by the length of the N-terminal 'matrix-targeting' sequence.     

The ionic strength of the intermembrane space of intact mitochondria is not affected by the pH or volume of the intermembrane space.

Ionic strength affects the electron transport activity of cytochrome c through its electrostatic interactions with redox partners and membrane lipids. We previously reported (Cortese, J.D., Voglino, A.L. and Hackenbrock, C.R. (1991) J. Cell Biol. 113, 1331-1340) that the ionic strength (I) of the intermembrane space (IMS-I) in isolated, intact condensed mitochondria is similar to the external, bulk I, over a wide range of bulk I. We now consider the possible effects of IMS-pH and IMS-volume, both variable parameters of mitochondrial function in situ, on IMS-I. IMS-pH and IMS-I were measured with pH- and I-sensitive fluorescent probes (highly fluorescent FITC-dextran for IMS-pH and FITC-BSA for IMS-I). These probes were delivered into the IMS of intact mitochondria via probe encapsulation into asolectin vesicles, followed by low pH-induced fusion of the vesicles with the outer membranes of intact mitochondria. IMS-pH was found to be 0.4-0.5 units lower than bulk pH over the pH range 6.0-8.5 for mitochondria with a large IMS-volume separating the two mitochondrial membranes (condensed configuration), and 0-0.2 units lower for mitochondria with a small IMS-volume and membranes closely opposed (orthodox configuration). This small pH difference between IMS-pM and bulk pH did not influence the similarity between IMS-I and bulk I. When the IMS-volume was osmotically decreased, bringing the two mitochondrial membranes in close proximity as in the orthodox configuration, IMS-I followed the bulk I above 10 mM but did not respond to changes in bulk I below 10 mM. The lack of response of the IMS-I below 10 mM indicates that the close proximity of the two mitochondrial membranes excludes ions only at low, nonphysiological I. Since the similarity of IMS-I and bulk I is unaffected by either IMS-pH or IMS-volume above a bulk I of 10 mM, at cytosolic physiological I (i.e., 100-150 mM) cytochrome c can be expected to be a free, three-dimensional diffusant in the IMS irrespective of the pH or volume of the IMS.     

Successive translocation into and out of the mitochondrial matrix: targeting of proteins to the intermembrane space by a bipartite signal peptide

We investigated the import and sorting pathways of cytochrome b2 and cytochrome c1, which are functionally located in the intermembrane space of mitochondria. Both proteins are synthesized on cytoplasmic ribosomes as larger precursors and are processed in mitochondria in two steps upon import. The precursors are first translocated across both mitochondrial membranes via contact sites into the matrix. Processing by the matrix peptidase leads to intermediate-sized forms, which are subsequently redirected across the inner membrane. The second proteolytic processing occurs in the intermembrane space. We conclude that the hydrophobic stretches in the presequences of the intermediate-sized forms do not stop transfer across the inner membrane, but rather act as transport signals to direct export from the matrix into the intermembrane space.     

Functional analysis of the domains in Cox11

Cox11 is an intrinsic mitochondrial membrane protein essential for the assembly of an active cytochrome c oxidase complex. Cox11 is tethered to the mitochondrial inner membrane by a single transmembrane helix. Domain mapping was carried out to determine the functional segments of the Cox11 protein. The C-terminal 189 residue Cu(I)-binding domain is shown to be exposed within the mitochondrial intermembrane space. This orientation was demonstrated by the proteolytic susceptibility of a C-terminal Myc epitope tag in mitoplasts but not intact mitochondria. Fusion of the N terminus of Cox11 to the matrix ribosomal protein Rsm22 results in a functional protein capable of suppressing the respiratory defect of both Deltacox11 cells and Deltarsm22 cells. The functionality of the fusion protein suggests that the Cox11 N terminus projects into the matrix. The fusion of the C-terminal segment of Cox11 to Rsm22 resembles a naturally occurring fusion of Cox11 in Schizosaccharomyces pombe to a sequence homologous to the Saccharomyces cerevisiae Rsm22. Studies on a series of SCO1/COX11 chimeras reveal that the matrix domain of Cox11 lacks a specific function, whereas the Cu(I) binding/donating function requires the yeast Cox11 sequence. The Cu(I)-binding domain from human Cox11 cannot functionally replace the yeast sequence. The copper domain of Cox11 may be an important docking motif for Cox1 or a Cox1-associated protein.