Enzymatic coupling of cholesterol intermediates to a mating pheromone precursor and to the ras protein.

The post-translational processing of the yeast a-mating pheromone precursor, Ras proteins, nuclear lamins, and some subunits of trimeric G proteins requires a set of complex modifications at their carboxyl termini. This processing includes three steps: prenylation of a cysteine residue, proteolytic processing, and carboxymethylation. In the yeast Saccharomyces cerevisiae, the product of the DPR1-RAM1 gene participates in this type of processing. Through the use of an in vitro assay with peptide substrates modeled after a presumptive a-mating pheromone precursor, it was discovered that mutations in DPR1-RAM1 cause a defect in the prenylation reaction. It was further shown that DPR1-RAM1 encodes an essential and limiting component of a protein prenyltransferase. These studies also implied a fixed order of the three processing steps shared by prenylated proteins: prenylation, proteolysis, then carboxymethylation. Because the yeast protein prenyltransferase could also prenylate human H-ras p21 precursor, the human DPR1-RAM1 analogue may be a useful target for anticancer chemotherapy.

Differential effectiveness of yeast cytochrome c oxidase subunit genes results from differences in expression not function.

In Saccharomyces cerevisiae, COX5a and COX5b encode two distinct forms of cytochrome c oxidase subunit V, Va and Vb, respectively. To determine the relative contribution of COX5a and COX5b to cytochrome c oxidase function, we have disrupted each gene. Cytochrome c oxidase activity levels and respiration rates of strains carrying null alleles of COX5a or COX5b or both indicate that some form of subunit V is required for cytochrome c oxidase function and that COX5a is much more effective than COX5b in providing this function. Wild-type respiration is supported by a single copy of either COX5a or COX5ab (a constructed chimeric gene sharing 5' sequences with COX5a). In contrast, multiple copies of COX5b or COX5ba (a chimeric gene with 5' sequences from COX5b) are required to support wild-type respiration. These results suggest that the decreased effectiveness of COX5b is due to inefficiency in gene expression rather than to any deficiency in the gene product, Vb. This conclusion is supported by two observations: (i) a COX5a-lacZ fusion gene produces more beta-galactosidase than a COX5b-lacZ fusion gene, and (ii) the COX5a transcript is significantly more abundant than the COX5b transcript or the COXsba transcript. We conclude that COX5a is expressed more efficiently than COX5b and that, although mature subunits Va and Vb are only 67% homologous, they do not differ significantly in their ability to assemble and function as subunits of the holoenzyme.

Genetic evidence for in vivo cross-specificity of the CaaX-box protein prenyltransferases farnesyltransferase and geranylgeranyltransferase-I in Saccharomyces cerevisiae.

Two protein prenyltransferase enzymes, farnesyltransferase (FTase) and geranylgeranyltransferase-I (GGTase-I), catalyze the covalent attachment of a farnesyl or geranylgeranyl lipid group to the cysteine of a CaaX sequence (cysteine [C], two aliphatic amino acids [aa], and any amino acid [X]. In vitro studies reported here confirm previous reports that CaaX proteins with a C-terminal serine are farnesylated by FTase and those with a C-terminal leucine are geranylgeranylated by GGTase-I. In addition, we found that FTase can farnesylate CaaX proteins with a C-terminal leucine and can transfer a geranylgeranyl group to some CaaX proteins. Genetic data indicate that FTase and GGTase-I have the same substrate preferences in vivo as in vitro and also show that each enzyme can prenylate some of the preferred substrates of the other enzyme in vivo. Specifically, the viability of yeast cells lacking FTase is due to prenylation of Ras proteins by GGTase-I. Although this GGTase-I dependent prenylation of Ras is sufficient for growth, it is not sufficient for mutationally activated Ras proteins to exert deleterious effects on growth. The dependence of the activated Ras phenotype on FTase can be bypassed by replacing the C-terminal serine with leucine. This altered form of Ras appears to be prenylated by both GGTase-I and FTase, since it produces an activated phenotype in a strain lacking either FTase or GGTase-I. Yeast cells can grow in the absence of GGTase-I as long as two essential substrates are overexpressed, but their growth is slow. Such strains are dependent on FTase for viability and are able to grow faster when FTase is overproduced, suggesting that FTase can prenylate the essential substrates of GGTase-I when they are overproduced.

Differential regulation of the two genes encoding Saccharomyces cerevisiae cytochrome c oxidase subunit V by heme and the HAP2 and REO1 genes.

In Saccharomyces cerevisiae, the COX5a and COX5b genes encode two forms of cytochrome c oxidase subunit V, Va and Vb. We report here that heme increases COX5a expression and decreases COX5b expression and that the HAP2 and REO1 genes are involved in positive regulation of COX5a and negative regulation of COX5b, respectively. Heme regulation of COX5a and COX5b may dictate which subunit V isoform is available for assembly into cytochrome c oxidase under conditions of high- and low-oxygen tension.

Structural analysis of two genes encoding divergent forms of yeast cytochrome c oxidase subunit V.

In Saccharomyces cerevisiae, subunit V of the inner mitochondrial membrane protein complex cytochrome c oxidase is encoded by two nonidentical genes, COX5a and COX5b. Both genes are present as single copies in S. cerevisiae and in several other Saccharomyces species. Nucleotide sequencing studies with the S. cerevisiae COX5 genes reveal that they encode proteins of 153 and 151 amino acids, respectively. Overall, the coding sequences of COX5a and COX5b have nucleotide and protein homologies of 67 and 66%, respectively. They are saturated for nucleotide substitutions that result in a synonomous codon, indicating a long divergence time between these two genes. Nucleotide sequences flanking the COX5a and COX5b coding regions exhibit no significant homology. The COX5a protein, pre-subunit Va, contains a 20-amino-acid leader peptide, whereas the COX5b protein, pre-subunit Vb, contains a 17-amino-acid leader peptide. These two leader peptides exhibit only 45% homology in the primary sequence, but have similar predicted secondary structures. By analyzing the RNA transcripts from both genes we have found that COX5a is a contiguous gene but that COX5b contains an intron. Surprisingly, the COX5b intron interrupts the AUG codon that initiates translation of the pre-subunit Vb polypeptide and contains a 5' donor splice sequence that differs from that normally found in yeast introns.

Plant farnesyltransferase can restore yeast Ras signaling and mating.

Farnesyltransferase (FTase) is a heterodimeric enzyme that modifies a group of proteins, including Ras, in mammals and yeasts. Plant FTase alpha and beta subunits were cloned from tomato and expressed in the yeast Saccharomyces cerevisiae to assess their functional conservation in farnesylating Ras and a-factor proteins, which are important for cell growth and mating. The tomato FTase beta subunit (LeFTB) alone was unable to complement the growth defect of ram1 delta mutant yeast strains in which the chromosomal FTase beta subunit gene was deleted, but coexpression of LeFTB with the plant alpha subunit gene (LeFTA) restored normal growth, Ras membrane association, and mating. LeFTB contains a novel 66-amino-acid sequence domain whose deletion reduces the efficiency of tomato FTase to restore normal growth to yeast ram1 delta strains. Coexpression of LeFTA and LeFTB in either yeast or insect cells yielded a functional enzyme that correctly farnesylated CaaX-motif-containing peptides. Despite their low degree of sequence homology, yeast and plant FTases shared similar in vivo and in vitro substrate specificities, demonstrating that this enzymatic modification of proteins with intermediates from the isoprenoid biosynthesis pathway is conserved in evolutionarily divergent eukaryotes.

The CaaX proteases, Afc1p and Rce1p, have overlapping but distinct substrate specificities.

Many proteins that contain a carboxyl-terminal CaaX sequence motif, including Ras and yeast a-factor, undergo a series of sequential posttranslational processing steps. Following the initial prenylation of the cysteine, the three C-terminal amino acids are proteolytically removed, and the newly formed prenylcysteine is carboxymethylated. The specific amino acids that comprise the CaaX sequence influence whether the protein can be prenylated and proteolyzed. In this study, we evaluated processing of a-factor variants with all possible single amino acid substitutions at either the a(1), the a(2), or the X position of the a-factor Ca(1)a(2)X sequence, CVIA. The substrate specificity of the two known yeast CaaX proteases, Afc1p and Rce1p, was investigated in vivo. Both Afc1p and Rce1p were able to proteolyze a-factor with A, V, L, I, C, or M at the a(1) position, V, L, I, C, or M at the a(2) position, or any amino acid at the X position that was acceptable for prenylation of the cysteine. Eight additional a-factor variants with a(1) substitutions were proteolyzed by Rce1p but not by Afc1p. In contrast, Afc1p was able to proteolyze additional a-factor variants that Rce1p may not be able to proteolyze. In vitro assays indicated that farnesylation was compromised or undetectable for 11 a-factor variants that produced no detectable halo in the wild-type AFC1 RCE1 strain. The isolation of mutations in RCE1 that improved proteolysis of a-factor-CAMQ, indicated that amino acid substitutions E139K, F189L, and Q201R in Rce1p affected its substrate specificity.

Removal of tropomyosin overlap and the co-operative response to increasing calcium concentrations of the acto-subfragment-1 ATPase.

The co-operative response of regulated actomyosin ATPase to increasing concentrations of calcium has been attributed to nearest-neighbor interactions, presumably between troponin-tropomyosin complexes. The degree of co-operativity was not decreased after the carboxy-terminal 11 amino acid residues had been removed from tropomyosin by carboxypeptidase A. This indicates that the interactions between neighboring troponin-tropomyosin complexes do not occur through the overlapping tropomyosin ends.

Identification of REO1, a gene involved in negative regulation of COX5b and ANB1 in aerobically grown Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the COX5a and COX5b genes constitute a small gene family that encodes two forms of cytochrome c oxidase subunit V, Va and Vb, either of which can provide a function essential for cytochrome c oxidase activity and respiration. In aerobically grown wild-type yeast cells, Va is the predominant form of subunit V. The COX5b gene alone does not produce enough Vb to support a respiration rate sufficient to allow growth on nonfermentable carbon sources. By selecting for mutations that increase the respiratory capacity of a strain deleted for COX5a, we have identified a gene that is involved in negative regulation of COX5b expression under aerobic growth conditions. Each of four independently isolated reo1 mutations are shown to be recessive, unlinked to COX5b, but dependent on COX5b for phenotypic expression. The mutations define a single complementation and linkage group: designated as REO1 for regulator of expression of oxidase. reo1 mutations increase expression of COX5b in aerobically grown cells, but not in anaerobically grown cells, where expression is already elevated. These mutations have no effect on COX5a, the other member of this small gene family which is positively regulated by heme and oxygen. The REO1 gene does play a role in repression of ANB1, a gene that is normally repressed under aerobic but not anaerobic conditions. Neither rox1 or rox3 mutations, which have previously been shown to increase ANB1 expression, are in the same complementation group as reo1 mutations.

Substrate specificity determinants in the farnesyltransferase beta-subunit.

Protein prenyltransferases catalyze the covalent attachment of isoprenoid lipids (farnesyl or geranylgeranyl) to a cysteine near the C terminus of their substrates. This study explored the specificity determinants for interactions between the farnesyltransferase of Saccharomyces cerevisiae and its protein substrates. A series of substitutions at amino acid 149 of the farnesyltransferase beta-subunit were tested in combination with a series of substitutions at the C-terminal amino acid of CaaX protein substrates Ras2p and a-factor. Efficient prenylation was observed when oppositely charged amino acids were present at amino acid 149 of the yeast farnesyltransferase beta-subunit and the C-terminal amino acid of the CaaX protein substrate, but not when like charges were present at these positions. This evidence for electrostatic interaction between amino acid 149 and the C-terminal amino acid of CaaX protein substrates leads to the prediction that the C-terminal amino acid of the protein substrate binds near amino acid 149 of the yeast farnesyltransferase beta-subunit.

The effect of low ATP concentrations on relaxation in the myosin regulated myofibrils from scallop.

Troponin-tropomyosin-regulated myofibrils show a significant increase in ATPase activity and contract in the absence of calcium when the ATP concentration falls significantly below the saturation level. By contrast, the ATPase of the myosin-regulated myofibrils of scallop striated muscle was not activated in the absence of calcium when the ATP concentration was lowered to 10mM. Nevertheless, a very small fraction of crossbridges were active at 10mM ATP resulting in very slow myofibrillar shortening. In contrast to the behaviour of rabbit contractile proteins there was no correlation between myofibrillar shortening and ATP induced turbidity changes of actomyosin taken from scallop.

Two nonidentical forms of subunit V are functional in yeast cytochrome c oxidase.

In Saccharomyces cerevisiae, the inner mitochondrial membrane protein cytochrome c oxidase is composed of nine polypeptide subunits. Six of these subunits (IV, V, VI, VII, VIIa, VIII) are encoded by the nuclear genome, and the remaining three (I, II, III) are encoded by mitochondrial DNA. We report here the existence of two nonidentical subunit V polypeptides, which are encoded by separate genes within the yeast genome. One gene, COX5a, encodes the polypeptide Va, normally found in preparations of holocytochrome c oxidase. The other gene, COX5b, encodes the polypeptide Vb, which cross-reacts with anti-subunit Va antiserum and restores respiratory competency and cytochrome oxidase activity in transformants of cox5a structural gene mutants. This polypeptide also copurifies with the holoenzyme prepared from these transformants. We have found that COX5b is expressed in vegetatively growing yeast cells, and that the Vb polypeptide can be detected in mitochondria from strain JM28, a cox5a mutant. This mutant has 15%-20% residual cytochrome oxidase activity, and it respires at 10%-15% the wild-type rate. By disrupting the COX5b gene in this strain, we show that this residual activity is directly attributable to the presence of a chromosomal copy of the COX5b gene. Taken together, these results suggest that Va or Vb can function as cytochrome oxidase subunits in yeast and that Vb may be used under some specific, as yet undefined, physiological conditions.

Potentiated state of the tropomyosin actin filament and nucleotide-containing myosin subfragment 1.

The main purpose of this study was to determine whether potentiation of acto-S-1 ATPase activity (activity higher than that obtained with tropomyosin-free actin) could be caused by nucleotide-containing acto-S-1 complexes. In addition, we wanted to know whether these complexes also have a positive cooperative effect on their own apparent binding constant under conditions where nucleotide-free acto-S-1 complexes cause potentiation of ATPase activity. Using calcium-saturated troponin-tropomyosin actin filaments, we observed potentiation of ATPase activity in the presence of 5.0 mM magnesium 5'-adenylyl imidodiphosphate (MgAMPPNP) and calculated that the ability of acto-S-1-AMPPNP complexes to cause potentiation must have been very similar to that of nucleotide-free acto-S-1 complexes. In extension of earlier studies, potentiated acto-S-1 ATPase activity was characterized by an increase in Vmax and, as observed before, a lowering of the apparent Km for subfragment 1 (S-1). Under conditions similar to those that produce the potentiation of acto-S-1 ATPase activity, the apparent actin binding constant of nucleotide-free S-1 was increased about 3-5 fold while the apparent binding constant of AMPPNP to actin-bound S-1 was reduced to (2.5-10) x 10(2) M-1 compared to that of about (1-5) x 10(3) M-1 for S-1 bound to tropomyosin-free actin. Under the same conditions, the apparent binding constant of S-1-AMPPNP to actin was not increased. We suggest that a potentiated state of the tropomyosin actin filament is produced by the cooperative action of acto-S-1 or acto-S-1-AMPPNP complexes. The potentiated state is characterized by an increase in the Vmax of the acto-S-1 ATPase activity, increased binding constants for S-1 and S-1-ADP, and increased binding of tropomyosin to actin.

Functional analysis of mitochondrial protein import in yeast.

In order to facilitate studies on protein localization to and sorting within yeast mitochondria, we have designed an experimental system that utilizes a new vector and a functional assay. The vector, which we call an LPS plasmid (for leader peptide substitution), employs a yeast COX5a gene (the structural gene for subunit Va of the inner membrane protein complex cytochrome c oxidase) as a convenient reporter for correct mitochondrial localization. Using in vitro mutagenesis, we have modified COX5a so that the DNA sequences encoding the wild-type subunit Va leader peptide can be precisely deleted and replaced with a given test sequence. The substituted leader peptide can then be analyzed for its ability to direct subunit Va to the inner mitochondrial membrane (to target and sort) by complementation or other in vivo assays. In this study we have tested the ability of several heterologous sequences to function in this system. The results of these experiments indicate that a functional leader peptide is required to target subunit Va to mitochondria. In addition, leader peptides, or portions thereof, derived from proteins located in other mitochondrial compartments can also be used to properly localize this polypeptide. The results presented here also indicate that the information necessary to sort subunit Va to the inner mitochondrial membrane does not reside in the leader peptide but rather in the mature subunit Va sequence.