Abhijit Mukhopadhyay

A co-translational model to explain the in vivo import of proteins into HeLa cell mitochondria

The dual signal approach, i.e. a mitochondrial signal at the N-terminus and an ER (endoplasmic reticulum) or a peroxisomal signal at the C-terminus of EGFP (enhanced green fluorescent protein), was employed in transfected HeLa cells to test for a co-translational import model. The signal peptide from OTC (ornithine transcarbamylase) or arginase II was fused to the N-terminus of EGFP, and an ER or peroxisomal signal was fused to its C-terminus. The rationale was that if the free preprotein remained in the cytosol, it could be distributed between the two organelles by using a post-translational pathway. The resulting fusion proteins were imported exclusively into mitochondria, suggesting that co-translational import occurred. Native preALDH (precursor of rat liver mitochondrial aldehyde dehydrogenase), preOTC and rhodanese, each with the addition of a C-terminal ER or peroxisomal signal, were also translocated only to the mitochondria, again showing that a co-translational import pathway exists for these native proteins. Import of preALDH(sp)-DHFR, a fusion protein consisting of the leader sequence (signal peptide) of preALDH fused to DHFR (dihydrofolate reductase), was studied in the presence of methotrexate, a substrate analogue for DHFR. It was found that 70% of the preALDH(sp)-DHFR was imported into mitochondria in the presence of methotrexate, implying that 70% of the protein utilized the co-translational import pathway and 30% used the post-translational import pathway. Thus it appears that co-translational import is a major pathway for mitochondrial protein import. A model is proposed to explain how competition between binding factors could influence whether or not a cytosolic carrier protein, such as DHFR, uses the co- or post-translational import pathway.

Bacterial signal peptide recognizes HeLa cell mitochondrial import receptors and functions as a mitochondrial leader sequence

Abstract.Phage display was used to identify new components of the mammalian mitochondrial receptor complex using Tom20 as a binding partner. Two peptides were identified. One had partial identity (SMLTVMA) with a bacterial signal peptide from Toho-1, a periplasmic protein. The other had partial identity with a mitochondrial inner membrane glutamate carrier. The bacterial signal peptide could carry a protein into mitochondria both in vivo and in vitro. The first six residues of the sequence, SMLTVM, were necessary for import but the two adjacent arginine residues in the 30-amino-acid leader were not critical for import. The signal peptides of Escherichia coli β-lactamase and Bacillsus subtilis lipase could not carry proteins into mitochondria. Presumably, the Toho-1 leader can adopt a structure compatible for recognition by the import apparatus.

Precursor Protein Is Readily Degraded in Mitochondrial Matrix Space if the Leader Is Not Processed by Mitochondrial Processing Peptidase

It is not known why leader peptides are removed by the mitochondrial processing peptidase after import into the matrix space. The leaders of yeast aldehyde dehydrogenase (pALDH) and malate dehydrogenase were mutated so that they would not be processed after import. The recombinant nonprocessed precursor of yeast pALDH possessed a similar specific activity as the corresponding mature form but was much less stable. The nonprocessed pALDH was transformed into a yeast strain missing ALDHs. The transformed yeast grew slowly on ethanol as the sole carbon source showing that the nonprocessed precursor was functional in vivo. Western blot analysis showed that the amount of precursor was 15-20% of that found in cells transformed with the native enzyme. Pulse-chase experiments revealed that the turnover rate for the nonprocessed precursor was greater than that of the mature protein indicating that the nonprocessed precursor could have been degraded. By using carbonyl cyanide m-chlorophenylhydrazone, we showed that the nonprocessed precursor was degraded in the matrix space. The nonprocessed precursor forms of precursor yeast malate dehydrogenase and rat liver pALDH also were degraded in the matrix space of HeLa cell mitochondria faster than their corresponding mature forms. In the presence of o-phenanthroline, an inhibitor of mitochondrial processing peptidase, the wild type precursor was readily degraded in the matrix space. Collectively, this study showed that the precursor form is less stable in the matrix space than is the mature form and provides an explanation for why the leader peptide is removed from the precursors.

In vitro evidence of inhibition of mitochondrial protease processing by HIV-1 protease inhibitors in yeast: a possible contribution to lipodystrophy syndrome.

Highly active antiretroviral therapy has been associated with the emergence of lipodystrophy syndromes that have clinical features commonly seen in patients with mitochondrial dysfunction. The effect of therapeutic protease inhibitors (PIs) on mitochondrial function is unknown. Mitochondrial matrix space proteins possess an amino-terminal leader peptide that is removed by the mitochondrial processing protease (MPP). Lack of cleavage could result in non- or dysfunctional mitochondrial proteins. The effects of different PIs on protease processing using pure MPP or yeast mitochondria, recognized models for mammalian counterparts, were examined in vitro. Multiple PIs were found to inhibit MPP, evidenced by accumulation of immature pALDH and decreased levels of processed ALDH. Both indinavir and amprenavir at 5.0 mg/ml resulted in significant inhibition of MPP. Although inhibition of MPP was also observed with ritonavir and saquinavir, the inhibition was difficult to quantify due to background inhibition of MPP by DMSO that was required to solubilize the drugs for the in vitro studies. Indinavir was also shown to inhibit MPP within yeast mitochondria. Lack of processing may impair mitochondrial function and contribute to the observed mitochondrial dysfunctions in patients receiving HAART and implicated in antiretroviral-associated lipodystrophy.

Timing and structural consideration for the processing of mitochondrial matrix space proteins by the mitochondrial processing peptidase (MPP)

Most mitochondrial matrix space proteins are synthesized as a precursor protein, and the N‐terminal extension of amino acids that served as the leader sequence is removed after import by the action of a metalloprotease called mitochondrial processing peptidase (MPP). The crystal structure of MPP has been solved very recently, and it has been shown that synthetic leader peptides bind with MPP in an extended conformation. However, it is not known how MPP recognizes hundreds of leader peptides with different primary and secondary structures or when during import the leader is removed. Here we took advantage of the fact that the structure of the leader from rat liver aldehyde dehydrogenase has been determined by 2D‐NMR to possess two helical portions separated by a three amino acid (RGP) linker. When the linker was deleted, the leader formed one long continuous helix that can target a protein to the matrix space but is not removed by the action of MPP. Repeats of two and three leaders were fused to the precursor protein to determine the stage of import at which processing occurs, if MPP could function as an endo peptidase, and if it would process if the cleavage site was part of a helix. Native or linker deleted constructs were used. Import into isolated yeast mitochondria or processing with recombinantly expressed MPP was performed. It was concluded that processing did not occur as the precursor was just entering the matrix space, but most likely coincided with the folding of the protein. Further, finding that hydrolysis could not take place if the processing site was part of a stable helix is consistent with the crystal structure of MPP. Lastly, it was found that MPP could function at sites as far as 108 residues from the N terminus of the precursor protein, but its ability to process decreases exponentially as the distance increases.

A point mutation produced a class 3 aldehyde dehydrogenase with increased protective ability against the killing effect of cyclophosphamide

Cyclophosphamides are pro-drugs whose killing agent is produced from an aldehyde that is formed by the action of a P450 oxidation step. The mustard from the aldehyde can destroy bone marrow cells as well as the tumor. Aldehyde dehydrogenase (EC 1.2.1.3) can oxidize the aldehyde and hence inactivate the cytotoxic intermediate but bone marrow has little, if any, of the enzyme. Others have shown that over-expression of the enzyme can afford protection of the marrow. A T186S mutant of the human stomach enzyme (ALDH3) that we developed has increased activity against the aldehyde compared to the native enzyme and HeLa cells transformed with the point mutant are better protected against the killing effect of the drug. It took threefold more drug to kill 90% of the cells transformed with the mutant compared to the native enzyme (15.8 compared to 5.1mM of a precursor of the toxic aldehyde). Analysis of molecular models makes it appear that removing the methyl group of threonine in the T186S mutant allows the bulky aldehyde to bind better. The mutant was found to be a poorer enzyme when small substrates such as benzaldehyde derivatives were investigated. Thus, the enzyme appears to be better only with large substrates such as the one produced by cyclophosphamide.

Binding of mitochondrial leader sequences to Tom20 assessed using a bacterial two‐hybrid system shows that hydrophobic interactions are essential and that some mutated leaders that do not bind Tom20 can still be imported

Previous studies pointed to the importance of leucine residues in the binding of mitochondrial leader sequences to Tom20, an outer membrane protein translocator that initially binds the leader during import. A bacteria two‐hybrid assay was here employed to determine if this could be an alternative way to investigate the binding of leader to the receptor. Leucine to alanine and arginine to glutamine mutations were made in the leader sequence from rat liver aldehyde dehydrogenase (pALDH). The leucine residues in the C‐terminal of pALDH leader were found to be essential for TOM20 binding. The hydrophobic residues of another mitochondrial leader F1β‐ATPase that were important for Tom20 binding were found at the C‐terminus of the leader. In contrast, it was the leucines in the N‐terminus of the leader of ornithine transcarbamylase that were essential for binding. Modeling the peptides to the structure of Tom20 showed that the hydrophobic residues from the three proteins could all fit into the hydrophobic binding pocket. The mutants of pALDH that did not bind to Tom20 were still imported in vivo in transformed HeLa cells or in vitro into isolated mitochondria. In contrast, the mutant from pOTC was imported less well (∼50%) while the mutant from F1β‐ATPase was not imported to any measurable extent. Binding to Tom20 might not be a prerequisite for import; however, it also is possible that import can occur even if binding to a receptor component is poor, so long as the leader binds tightly to another component of the translocator.

Mitochondrial NAD dependent aldehyde dehydrogenase either from yeast or human replaces yeast cytoplasmic NADP dependent aldehyde dehydrogenase for the aerobic growth of yeast on ethanol.

BACKGROUND In a previous study, we deleted three aldehyde dehydrogenase (ALDH) genes, involved in ethanol metabolism, from yeast Saccharomyces cerevisiae and found that the triple deleted yeast strain did not grow on ethanol as sole carbon source. The ALDHs were NADP dependent cytosolic ALDH1, NAD dependent mitochondrial ALDH2 and NAD/NADP dependent mitochondrial ALDH5. Double deleted strain ΔALDH2+ΔALDH5 or ΔALDH1+ΔALDH5 could grow on ethanol. However, the double deleted strain ΔALDH1+ΔALDH2 did not grow in ethanol. METHODS Triple deleted yeast strain was used. Mitochondrial NAD dependent ALDH from yeast or human was placed in yeast cytosol. RESULTS In the present study we found that a mutant form of cytoplasmic ALDH1 with very low activity barely supported the growth of the triple deleted strain (ΔALDH1+ΔALDH2+ΔALDH5) on ethanol. Finding the importance of NADP dependent ALDH1 on the growth of the strain on ethanol we examined if NAD dependent mitochondrial ALDH2 either from yeast or human would be able to support the growth of the triple deleted strain on ethanol if the mitochondrial form was placed in cytosol. We found that the NAD dependent mitochondrial ALDH2 from yeast or human was active in cytosol and supported the growth of the triple deleted strain on ethanol. CONCLUSION This study showed that coenzyme preference of ALDH is not critical in cytosol of yeast for the growth on ethanol. GENERAL SIGNIFICANCE The present study provides a basis to understand the coenzyme preference of ALDH in ethanol metabolism in yeast.