Bovine coupling factor 6, with just 14.5% shared identity, replaces subunit h in the yeast ATP synthase.

The mammalian mitochondrial ATP synthase is composed of at least 16 polypeptides. With the exception of coupling factor F(6), there are likely yeast homologs for each of these polypeptides. There are no obvious yeast homologs of F(6), as predicted from primary sequence comparison of the putative peptides encoded by the open reading frames in the yeast genome. In this manuscript, we demonstrate that expression of bovine F(6) complements a null mutant in ATP14 gene in yeast Saccharomyces cerevisiae. Subunit h of the yeast ATP synthase is encoded by ATP14 and is just 14.5% identical to bovine F(6). Expression of bovine F(6) in an atp14 null mutant strain recovers oxidative phosphorylation, and the ATP synthase is active, although functioning with a lower efficiency than the wild type enzyme. Like subunit h, bovine F(6) is shown to interact mainly with subunit 4 (subunit b), a component of the second stalk of the enzyme. These data indicated the subunit h is the yeast homolog of mammalian coupling factor F(6).

Complementation of deletion mutants in the genes encoding the F1-ATPase by expression of the corresponding bovine subunits in yeast S. cerevisiae.

The F1F0 ATP synthase is composed of the F1-ATPase which is bound to F0, in the inner membrane of the mitochondrion. Assembly and function of the enzyme is a complicated task requiring the interactions of many proteins for the folding, import, assembly, and function of the enzyme. The F1-ATPase is a multimeric enzyme composed of five subunits in the stoichiometry of alpha3beta3gammadeltaepsilon. This study demonstrates that four of the five bovine subunits of the F1-ATPase can be imported and function in an otherwise yeast enzyme effectively complementing mutations in the genes encoding the corresponding yeast ATPase subunits. In order to demonstrate this, the coding regions of each of the five genes were separately deleted in yeast providing five null mutant strains. All of the strains displayed negative or a slow growth phenotype on medium containing glycerol as the carbon source and strains with a null mutation in the gene encoding the gamma-, delta- or epsilon-gene became completely, or at a high frequency, cytoplasmically petite. The subunits of bovine F1 were expressed individually in the yeast strains with the corresponding null mutations and targeted to the mitochondrion using a yeast mitochondrial leader peptide. Expression of the bovine alpha-, beta-, gamma-, and epsilon-, but not the delta-, subunit complemented the corresponding null mutations in yeast correcting the corresponding negative phenotypes. These results indicate that yeast is able to import, assemble subunits of bovine F1-ATPase in mitochondria and form a functional chimeric yeast/bovine enzyme complex.

Structure/function of the beta-barrel domain of F1-ATPase in the yeast Saccharomyces cerevisiae.

The first 90 amino acids of the alpha- and beta-subunits of mitochondrial F1-ATPase are folded into beta-barrel domains and were postulated to be important for stabilizing the enzyme (Abrahams, J. P., Leslie, A. G., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628). The role of the domains was studied by making chimeric enzymes, replacing the domains from the yeast Saccharomyces cerevisiae enzyme with the corresponding domains from the enzyme of the thermophilic bacterium Bacillus PS3. The enzymes containing the chimeric alpha-, beta-, or alpha- and beta-subunits were not functional. However, gain-of-function mutations were obtained from the strain containing the enzyme with the chimeric PS3/yeast beta-subunit. The gain-of-function mutations were all in codons encoding the beta-barrel domain of the beta-subunit, and the residues appear to map out a region of subunit-subunit interactions. Gain-of-function mutations were also obtained that provided functional expression of the chimeric PS3/yeast alpha- and beta-subunits together. Biochemical analysis of this active chimeric enzyme indicated that it was not significantly more thermostable or labile than the wild type. The results of this study indicate that the beta-barrel domains form critical contacts (distinct from those between the alpha- and beta-subunits) that are important for the assembly of the ATP synthase.

Epistatic interactions of deletion mutants in the genes encoding the F1-ATPase in yeast Saccharomyces cerevisiae.

The F1-ATPase is a multimeric enzyme (alpha3 beta3 gamma delta epsilon) primarily responsible for the synthesis of ATP under aerobic conditions. The entire coding region of each of the genes was deleted separately in yeast, providing five null mutant strains. Strains with a deletion in the genes encoding alpha-, beta-, gamma- or delta-subunits were unable to grow, while the strain with a null mutation in epsilon was able to grow slowly on medium containing glycerol as the carbon source. In addition, strains with a null mutation in gamma or delta became 100% rho0/rho- and the strain with the null mutation in gamma grew much more slowly on medium containing glucose. These additional phenotypes were not observed in strains with the double mutations: Delta alpha Delta gamma, Delta beta Delta gamma, Deltaatp11 Delta gamma, Delta alpha Delta delta, Delta beta Delta delta or Deltaatp11 Delta delta. These results indicate that epsilon is not an essential component of the ATP synthase and that mutations in the genes encoding the alpha- and beta-subunits and in ATP11 are epistatic to null mutations in the genes encoding the gamma- and delta-subunits. These data suggest that the propensity to form rho0/rho- mutations in the gamma and delta null deletion mutant stains and the slow growing phenotypes of the null gamma mutant strain are due to the assembly of F1 deficient in the corresponding subunit. These results have profound implications for the physiology of normal cells.

Mutational analysis of the insulin-like growth factor I prohormone processing site.

Insulin-like growth factor I (IGF-I) is a mitogenic peptide that is produced in most tissues and cell lines and plays an important role in embryonic development and postnatal growth. IGF-I is initially synthesized as a prohormone precursor that is converted to mature IGF-I by endoproteolytic removal of the carboxyl-terminal E-domain. Regulation of the conversion of proIGF-I to mature IGF-I is a potential mechanism by which the biological activity of this growth factor might be modulated. Endoproteolysis of the IGF-I prohormone occurs at the unique pentabasic motif Lys-X-X-Lys-X-X-Arg71-X-X-Arg-X-X-Arg. Recently, a family of enzymes which cleave prohormone precursors at sites containing multiple basic residues has been discovered. The goals of this study were 1) to determine which basic residues in the pentabasic proIGF-I processing site were necessary for proper cleavage and 2) to examine the role that subtilisin-related proprotein convertase 1 (SPC1/furin) might play in proIGF-I processing. We have shown that an expression vector coding for an epitope-tagged proIGF-I directs synthesis and secretion of mature IGF-I-(1-70), extended IGF-I-(1-76), proIGF-I, and N-glycosylated proIGF-I in human embryonic kidney 293 cells. Extended IGF-I-(1-76) is produced by cleavage at Arg77 and requires both Arg74 (P4) and Arg77 (P1). Cleavage at Arg77 does not occur in the SPC1-deficient cell lines RPE.40 and LoVo, suggesting that processing at this site is mediated by SPC1. Mature IGF-I-(1-70) is produced by cleavage at Arg71 and requires both Lys68 (P4) and Arg71 (P1). Lys65 in the P7 position is important for efficient cleavage. SPC1 is not required for processing at Arg71 since this cleavage occurs in RPE.40 and LoVo cells. These data suggest the existence of a processing enzyme which is specific for the Lys-X-X-Arg motif of proIGF-I.