Roles of Phosphatidylethanolamine and of Its Several Biosynthetic Pathways in Saccharomyces cerevisiae

Three different pathways lead to the synthesis of phosphatidylethanolamine (PtdEtn) in yeast, one of which is localized to the inner mitochondrial membrane. To study the contribution of each of these pathways, we constructed a series of deletion mutants in which different combinations of the pathways are blocked. Analysis of their growth phenotypes revealed that a minimal level of PtdEtn is essential for growth. On fermentable carbon sources such as glucose, endogenous ethanolaminephosphate provided by sphingolipid catabolism is sufficient to allow synthesis of the essential amount of PtdEtn through the cytidyldiphosphate (CDP)-ethanolamine pathway. On nonfermentable carbon sources, however, a higher level of PtdEtn is required for growth, and the amounts of PtdEtn produced through the CDP-ethanolamine pathway and by extramitochondrial phosphatidylserine decarboxylase 2 are not sufficient to maintain growth unless the action of the former pathway is enhanced by supplementing the growth medium with ethanolamine. Thus, in the absence of such supplementation, production of PtdEtn by mitochondrial phosphatidylserine decarboxylase 1 becomes essential. In psd1Δ strains or cho1Δ strains (defective in phosphatidylserine synthesis), which contain decreased amounts of PtdEtn, the growth rate on nonfermentable carbon sources correlates with the content of PtdEtn in mitochondria, suggesting that import of PtdEtn into this organelle becomes growth limiting. Although morphological and biochemical analysis revealed no obvious defects of PtdEtn-depleted mitochondria, the mutants exhibited an enhanced formation of respiration-deficient cells. Synthesis of glycosylphosphatidylinositol-anchored proteins is also impaired in PtdEtn-depleted cells, as demonstrated by delayed maturation of Gas1p. Carboxypeptidase Y and invertase, on the other hand, were processed with wild-type kinetics. Thus, PtdEtn depletion does not affect protein secretion in general, suggesting that high levels of nonbilayer-forming lipids such as PtdEtn are not essential for membrane vesicle fusion processes in vivo.

Structural Requirements of Tom40 for Assembly into Preexisting TOM Complexes of Mitochondria

Tom40 is the major subunit of the translocase of the outer mitochondrial membrane (the TOM complex). To study the assembly pathway of Tom40, we have followed the integration of the protein into the TOM complex in vitro and in vivo using wild-type and altered versions of the Neurospora crassa Tom40 protein. Upon import into isolated mitochondria, Tom40 precursor proteins lacking the first 20 or the first 40 amino acid residues were assembled as the wild-type protein. In contrast, a Tom40 precursor lacking residues 41 to 60, which contains a highly conserved region of the protein, was arrested at an intermediate stage of assembly. We constructed mutant versions of Tom40 affecting this region and transformed the genes into a sheltered heterokaryon containing a tom40 null nucleus. Homokaryotic strains expressing the mutant Tom40 proteins had growth rate defects and were deficient in their ability to form conidia. Analysis of the TOM complex in these strains by blue native gel electrophoresis revealed alterations in electrophoretic mobility and a tendency to lose Tom40 subunits from the complex. Thus, both in vitro and in vivo studies implicate residues 41 to 60 as containing a sequence required for proper assembly/stability of Tom40 into the TOM complex. Finally, we found that TOM complexes in the mitochondrial outer membrane were capable of exchanging subunits in vitro. A model is proposed for the integration of Tom40 subunits into the TOM complex.

Role of the Ascorbate-Glutathione Cycle of Mitochondria and Peroxisomes in the Senescence of Pea Leaves1

We investigated the relationship between H2O2 metabolism and the senescence process using soluble fractions, mitochondria, and peroxisomes from senescent pea (Pisum sativum L.) leaves. After 11 d of senescence the activities of Mn-superoxide dismutase, dehydroascorbate reductase (DHAR), and glutathione reductase (GR) present in the matrix, and ascorbate peroxidase (APX) and monodehydroascorbate reductase (MDHAR) activities localized in the mitochondrial membrane, were all substantially decreased in mitochondria. The mitochondrial ascorbate and dehydroascorbate pools were reduced, whereas the oxidized glutathione levels were maintained. In senescent leaves the H2O2 content in isolated mitochondria and the NADH- and succinate-dependent production of superoxide (O2·−) radicals by submitochondrial particles increased significantly. However, in peroxisomes from senescent leaves both membrane-bound APX and MDHAR activities were reduced. In the matrix the DHAR activity was enhanced and the GR activity remained unchanged. As a result of senescence, the reduced and the oxidized glutathione pools were considerably increased in peroxisomes. A large increase in the glutathione pool and DHAR activity were also found in soluble fractions of senescent pea leaves, together with a decrease in GR, APX, and MDHAR activities. The differential response to senescence of the mitochondrial and peroxisomal ascorbate-glutathione cycle suggests that mitochondria could be affected by oxidative damage earlier than peroxisomes, which may participate in the cellular oxidative mechanism of leaf senescence longer than mitochondria.

Environmental and developmental signals modulate proline homeostasis: evidence for a negative transcriptional regulator.

In many plants, osmotic stress induces a rapid accumulation of proline through de novo synthesis from glutamate. This response is thought to play a pivotal role in osmotic stress tolerance [Kishor, P. B. K., Hong, Z., Miao, G.-H., Hu, C.-A. A. and Verma, D. P. S. (1995) Plant Physiol. 108, 1387-1394]. During recovery from osmotic stress, accumulated proline is rapidly oxidized to glutamate and the first step of this process is catalyzed by proline oxidase. We have isolated a full-length cDNA from Arabidopsis thaliana, At-POX, which maps to a single locus on chromosome 3 and that encodes a predicted polypeptide of 499 amino acids showing significant similarity with proline oxidase sequences from Drosophila and Saccharomyces cerevisiae (55.5% and 45.1%, respectively). The predicted location of the encoded polypeptide is the inner mitochondrial membrane. RNA gel blot analysis revealed that At-POX mRNA levels declined rapidly upon osmotic stress and this decline preceded proline accumulation. On the other hand, At-POX mRNA levels rapidly increased during recovery. Free proline, exogenously added to plants, was found to be an effective inducer of At-POX expression; indeed, At-POX was highly expressed in flowers and mature seeds where the proline level is higher relative to other organs of Arabidopsis. Our results indicate that stress- and developmentally derived signals interact to determine proline homeostasis in Arabidopsis.

Mitochondria are selective targets for the protective effects of heat shock against oxidative injury.

Heat shock (HS) proteins (HSPs) induce protection against a number of stresses distinct from HS, including reactive oxygen species. In the human premonocytic line U937, we investigated in whole cells the effects of preexposure to HS and exposure to hydrogen peroxide (H2O2) on mitochondrial membrane potential, mass, and ultrastructure. HS prevented H2O2-induced alterations in mitochondrial membrane potential and cristae formation while increasing expression of HSPs and the protein product of bcl-2. Protection correlated best with the expression of the 70-kDa HSP, hsp70. We propose that mitochondria represent a selective target for HS-mediated protection against oxidative injury.

Disruption of the gene for hsp30, an alpha-crystallin-related heat shock protein of Neurospora crassa, causes defects in thermotolerance.

The alpha-crystallin-related heat shock proteins are produced by all eukaryotes, but the role of these proteins in thermoprotection remains unclear. To investigate the function of one of these proteins, we disrupted expression of the single-copy hsp30 gene of Neurospora crassa, using repeat-induced point mutagenesis, and we generated and characterized mutant strains that were deficient in hsp30 synthesis. These strains could grow at high temperature and they acquired thermotolerance from a heat shock. However, the hsp30-defective strains proved to be extremely sensitive to the combined stresses of high temperature and carbohydrate limitation, enforced by the addition of a nonmetabolizable glucose analogue. Under these conditions, their survival was reduced by 90% compared with wild-type cells. This sensitive phenotype was reversed by reintroduction of a functional hsp30 gene into the mutant strains. The mutant cells contained mitochondria from which a 22-kDa protein was readily extracted with detergents, in contrast to its retention by the mitochondria of wild-type cells. Antibodies against hsp30 coimmunoprecipitated a protein also of approximately 22 kDa from wild-type cells. Results of this study suggest that hsp30 may be important for efficient carbohydrate utilization during high temperature stress and that it may interact with other mitochondrial membrane proteins and function as a protein chaperone.

Uncoupling of transfer of the presequence and unfolding of the mature domain in precursor translocation across the mitochondrial outer membrane

Translocation of mitochondrial precursor proteins across the mitochondrial outer membrane is facilitated by the translocase of the outer membrane (TOM) complex. By using site-specific photocrosslinking, we have mapped interactions between TOM proteins and a mitochondrial precursor protein arrested at two distinct stages, stage A (accumulated at 0°C) and stage B (accumulated at 30°C), in the translocation across the outer membrane at high resolution not achieved previously. Although the stage A and stage B intermediates were assigned previously to the forms bound to the cis site and the trans site of the TOM complex, respectively, the results of crosslinking indicate that the presequence of the intermediates at both stage A and stage B is already on the trans side of the outer membrane. The mature domain is unfolded and bound to Tom40 at stage B whereas it remains folded at stage A. After dissociation from the TOM complex, translocation of the stage B intermediate, but not of the stage A intermediate, across the inner membrane was promoted by the intermembrane-space domain of Tom22. We propose a new model for protein translocation across the outer membrane, where translocation of the presequence and unfolding of the mature domain are not necessarily coupled.

A Splice-Isoform of Vesicle-associated Membrane Protein-1 (VAMP-1) Contains a Mitochondrial Targeting Signal

Screening of a library derived from primary human endothelial cells revealed a novel human isoform of vesicle-associated membrane protein-1 (VAMP-1), a protein involved in the targeting and/or fusion of transport vesicles to their target membrane. We have termed this novel isoform VAMP-1B and designated the previously described isoform VAMP-1A. VAMP-1B appears to be an alternatively spliced form of VAMP-1. A similar rat splice variant of VAMP-1 (also termed VAMP-1B) has recently been reported. Five different cultured cell lines, from different lineages, all contained VAMP-1B but little or no detectable VAMP-1A mRNA, as assessed by PCR. In contrast, brain mRNA contained VAMP-1A but no VAMP-1B. The VAMP-1B sequence encodes a protein identical to VAMP-1A except for the carboxy-terminal five amino acids. VAMP-1 is anchored in the vesicle membrane by a carboxy-terminal hydrophobic sequence. In VAMP-1A the hydrophobic anchor is followed by a single threonine, which is the carboxy-terminal amino acid. In VAMP-1B the predicted hydrophobic membrane anchor is shortened by four amino acids, and the hydrophobic sequence is immediately followed by three charged amino acids, arginine-arginine-aspartic acid. Transfection of human endothelial cells with epitope-tagged VAMP-1B demonstrated that VAMP-1B was targeted to mitochondria whereas VAMP-1A was localized to the plasma membrane and endosome-like structures. Analysis of C-terminal mutations of VAMP-1B demonstrated that mitochondrial targeting depends both on the addition of positive charge at the C terminus and a shortened hydrophobic membrane anchor. These data suggest that mitochondria may be integrated, at least at a mechanistic level, to the vesicular trafficking pathways that govern protein movement between other organelles of the cell.

Molecular cloning of Ian4: a BCR/ABL-induced gene that encodes an outer membrane mitochondrial protein with GTP-binding activity

Using the representation difference analysis technique, we have identified a novel gene, Ian4, which is preferentially expressed in hematopoietic precursor 32D cells transfected with wild-type versus mutant forms of the Bcr/Abl oncogene. Ian4 expression was undetectable in 32D cells transfected with v-src, oncogenic Ha-ras or v-Abl. Murine Ian4 maps to chromosome 6, 25 cM from the centromere. The Ian4 mRNA contains two open reading frames (ORFs) separated by 5 nt. The first ORF has the potential to encode for a polypeptide of 67 amino acids without apparent homology to known proteins. The second ORF encodes a protein of 301 amino acids with a GTP/ATP-binding site in the N-terminus and a hydrophobic domain in the extreme C-terminus. The IAN-4 protein resides in the mitochondrial outer membrane and the last 20 amino acids are necessary for this localization. The IAN-4 protein has GTP-binding activity and shares sequence homology with a novel family of putative GTP-binding proteins: the immuno-associated nucleotide (IAN) family.

3T3 fibroblasts transfected with a cDNA for mitochondrial aspartate aminotransferase express plasma membrane fatty acid-binding protein and saturable fatty acid uptake.

To explore the relationship between mitochondrial aspartate aminotransferase (mAspAT; EC 2.6.1.1) and plasma membrane fatty acid-binding protein (FABPpm) and their role in cellular fatty acid uptake, 3T3 fibroblasts were cotransfected with plasmid pMAAT2, containing a full-length mAspAT cDNA downstream of a Zn(2+)-inducible metallothionein promoter, and pFR400, which conveys methotrexate resistance. Transfectants were selected in methotrexate, cloned, and exposed to increasing methotrexate concentrations to induce gene amplification. Stably transfected clones were characterized by Southern blotting; those with highest copy numbers of pFR400 alone (pFR400) or pFR400 and pMAAT2 (pFR400/pMAAT2) were expanded for further study. [3H]Oleate uptake was measured in medium containing 500 microM bovine serum albumin and 125-1000 microM total oleate (unbound oleate, 18-420 nM) and consisted of saturable and nonsaturable components. pFR400/pMAAT2 cells exhibited no increase in the rate constant for nonsaturable oleate uptake or in the uptake rate of [14C]octanoate under any conditions. By contrast, Vmax (fmol/sec per 50,000 cells) of the saturable oleate uptake component increased 3.5-fold in pFR400/pMAAT2 cells compared to pFR400, with a further 3.2-fold increase in the presence of Zn2+. Zn2+ had no effect in pFR400 controls (P > 0.5). The overall increase in Vmax between pFR400 and pFR400/pMAAT2 in the presence of Zn2+ was 10.4-fold (P < 0.01) and was highly correlated (r = 0.99) with expression of FABPpm in plasma membranes as determined by Western blotting. Neither untransfected 3T3 nor pFR400 cells expressed cell surface FABPpm detectable by immunofluorescence. By contrast, plasma membrane immunofluorescence was detected in pFR400/pMAAT2 cells, especially if cultured in 100 microM Zn2+. The data support the dual hypotheses that mAspAT and FABPpm are identical and mediate saturable long-chain free fatty acid uptake.

Measurement of cytosolic, mitochondrial, and Golgi pH in single living cells with green fluorescent proteins

Many cellular events depend on a tightly compartmentalized distribution of H+ ions across membrane-bound organelles. However, measurements of organelle pH in living cells have been scarce. Several mutants of the Aequorea victoria green fluorescent protein (GFP) displayed a pH-dependent absorbance and fluorescent emission, with apparent pKa values ranging from 6.15 (mutations F64L/S65T/H231L) and 6.4 (K26R/F64L/S65T/Y66W/N146I/M153T/V163A/N164H/H231L) to a remarkable 7.1 (S65G/S72A/T203Y/H231L). We have targeted these GFPs to the cytosol plus nucleus, the medial/trans-Golgi by fusion with galactosyltransferase, and the mitochondrial matrix by using the targeting signal from subunit IV of cytochrome c oxidase. Cells in culture transfected with these cDNAs displayed the expected subcellular localization by light and electron microscopy and reported local pH that was calibrated in situ with ionophores. We monitored cytosolic and nuclear pH of HeLa cells, and mitochondrial matrix pH in HeLa cells and in rat neonatal cardiomyocytes. The pH of the medial/trans-Golgi was measured at steady-state (calibrated to be 6.58 in HeLa cells) and after various manipulations. These demonstrated that the Golgi membrane in intact cells is relatively permeable to H+, and that Cl− serves as a counter-ion for H+ transport and likely helps to maintain electroneutrality. The amenability to engineer GFPs to specific subcellular locations or tissue targets using gene fusion and transfer techniques should allow us to examine pH at sites previously inaccessible.

Cloning and characterization of a plastidal and a mitochondrial isoform of tobacco protoporphyrinogen IX oxidase

Protoporphyrinogen IX oxidase is the last enzyme in the common pathway of heme and chlorophyll synthesis and provides precursor for the mitochondrial and plastidic heme synthesis and the predominant chlorophyll synthesis in plastids. We cloned two different, full-length tobacco cDNA sequences by complementation of the protoporphyrin-IX-accumulating Escherichia coli hemG mutant from heme auxotrophy. The two sequences show similarity to the recently published Arabidopsis PPOX, Bacillus subtilis hemY, and to mammalian sequences encoding protoporphyrinogen IX oxidase. One cDNA sequence encodes a 548-amino acid residues protein with a putative transit sequence of 50 amino acid residues, and the second cDNA encodes a protein of 504 amino acid residues. Both deduced protein sequences share 27.2% identical amino acid residues. The first in vitro translated protoporphyrinogen IX oxidase could be translocated to plastids, and the approximately 53-kDa mature protein was detected in stroma and membrane fraction. The second enzyme was targeted to mitochondria without any detectable reduction in size. Localization of both enzymes in subcellular fractions was immunologically confirmed. Steady-state RNA analysis indicates an almost synchronous expression of both genes during tobacco plant development, greening of young seedlings, and diurnal and circadian growth. The mature plastidal and the mitochondrial isoenzyme were overexpressed in E. coli. Bacterial extracts containing the recombinant mitochondrial enzyme exhibit high protoporphyrinogen IX oxidase activity relative to control strains, whereas the plastidal enzyme could only be expressed as an inactive peptide. The data presented confirm a compartmentalized pathway of tetrapyrrole synthesis with protoporphyrinogen IX oxidase in plastids and mitochondria.

The dimensions of the protein import channels in the outer and inner mitochondrial membranes

Most mitochondrial proteins are imported into mitochondria through transmembrane channels composed largely, and perhaps exclusively, of proteins. We have determined the effective internal diameter of the protein import channel in the mitochondrial outer membrane to be between 20 Å and 26 Å during translocation. The diameter of the import channel in the inner membrane is smaller than the diameter of the outer membrane import channel. These results were obtained by measuring the effect of rigid steric bulk introduced into precursor proteins on import.

A mammalian mitochondrial drug receptor functions as a bacterial “oxygen” sensor

The rat mitochondrial outer membrane-localized benzodiazepine receptor (MBR) was expressed in wild-type and TspO− (tryptophan-rich sensory protein) strains of the facultative photoheterotroph, Rhodobacter sphaeroides 2.4.1, and was shown to retain its structure within the bacterial outer membrane as assayed by its binding properties with a variety of MBR ligands. Functionally, it was able to substitute for TspO by negatively regulating the expression of photosynthesis genes in response to oxygen. This effect was reversed pharmacologically with the MBR ligand PK11195. These results suggest a close evolutionary and functional relationship between the bacterial TspO and the MBR. This relationship provides further support for the origin of the mammalian mitochondrion from a “photosynthetic” precursor. Finally, these findings provide novel insights into the physiological role that has been obscure for the MBR in situ.

Biogenesis of Tim Proteins of the Mitochondrial Carrier Import Pathway: Differential Targeting Mechanisms and Crossing Over with the Main Import Pathway

Two major routes of preprotein targeting into mitochondria are known. Preproteins carrying amino-terminal signals mainly use Tom20, the general import pore (GIP) complex and the Tim23–Tim17 complex. Preproteins with internal signals such as inner membrane carriers use Tom70, the GIP complex, and the special Tim pathway, involving small Tims of the intermembrane space and Tim22–Tim54 of the inner membrane. Little is known about the biogenesis and assembly of the Tim proteins of this carrier pathway. We report that import of the preprotein of Tim22 requires Tom20, although it uses the carrier Tim route. In contrast, the preprotein of Tim54 mainly uses Tom70, yet it follows the Tim23–Tim17 pathway. The positively charged amino-terminal region of Tim54 is required for membrane translocation but not for targeting to Tom70. In addition, we identify two novel homologues of the small Tim proteins and show that targeting of the small Tims follows a third new route where surface receptors are dispensable, yet Tom5 of the GIP complex is crucial. We conclude that the biogenesis of Tim proteins of the carrier pathway cannot be described by either one of the two major import routes, but involves new types of import pathways composed of various features of the hitherto known routes, including crossing over at the level of the GIP.