Domain interactions in the yeast ATP binding cassette transporter Ycf1p: intragenic suppressor analysis of mutations in the nucleotide binding domains.

The yeast cadmium factor (Ycf1p) is a vacuolar ATP binding cassette (ABC) transporter required for heavy metal and drug detoxification. Cluster analysis shows that Ycf1p is strongly related to the human multidrug-associated protein (MRP1) and cystic fibrosis transmembrane conductance regulator and therefore may serve as an excellent model for the study of eukaryotic ABC transporter structure and function. Identifying intramolecular interactions in these transporters may help to elucidate energy transfer mechanisms during transport. To identify regions in Ycf1p that may interact to couple ATPase activity to substrate binding and/or movement across the membrane, we sought intragenic suppressors of ycf1 mutations that affect highly conserved residues presumably involved in ATP binding and/or hydrolysis. Thirteen intragenic second-site suppressors were identified for the D777N mutation which affects the invariant Asp residue in the Walker B motif of the first nucleotide binding domain (NBD1). Two of the suppressor mutations (V543I and F565L) are located in the first transmembrane domain (TMD1), nine (A1003V, A1021T, A1021V, N1027D, Q1107R, G1207D, G1207S, S1212L, and W1225C) are found within TMD2, one (S674L) is in NBD1, and another one (R1415G) is in NBD2, indicating either physical proximity or functional interactions between NBD1 and the other three domains. The original D777N mutant protein exhibits a strong defect in the apparent affinity for ATP and V(max) of transport. The phenotypic characterization of the suppressor mutants shows that suppression does not result from restoring these alterations but rather from a change in substrate specificity. We discuss the possible involvement of Asp777 in coupling ATPase activity to substrate binding and/or transport across the membrane.

Yol082p, a novel CVT protein involved in the selective targeting of aminopeptidase I to the yeast vacuole.

The yeast vacuolar enzyme aminopeptidase I (API) is synthesized in the cytoplasm as a precursor (pAPI). Upon its assembly into dodecamers, pAPI is wrapped by double-membrane saccular structures for its further transport within vesicles that fuse with the vacuolar membrane and release their content in the vacuolar lumen. Targeting of API to the vacuole occurs by two alternative transport routes, the cvt and the autophagy pathways, which although mechanistically similar specifically operate under vegetative growth or nitrogen starvation conditions, respectively. We have studied the role of Yol082p, a protein identified by its ability to interact with API, in the transport of its precursor to the vacuole. We show that Yol082p interacts with mature API, an interaction that is strengthened by the amino extension of the API protein. Yol082p is required for targeting of pAPI to the vacuole, both under growing and short term nitrogen starvation conditions. Absence of Yol082p does not impede the assembly of pAPI into dodecamers, but precludes the enclosure of pAPI within transport vesicles. Microscopy studies show that during vegetative growth Yol082p is distributed between a cytoplasmic pool and a variable number of 0.13--0.27-microm round, mobile structures, which are no longer observed under conditions of nitrogen starvation, and become larger in cells expressing the inactive Yol082 Delta C32p, or lacking Apg12p. In contrast to the autophagy mutants involved in API transport, a Delta yol082 strain does not lose viability under nitrogen starvation conditions, indicating normal function of the autophagy pathway. The data are consistent with a role of Yol082p in an early step of the API transport, after its assembly into dodecamers. Because Yol082p fulfills the functional requisites that define the CVT proteins, we propose to name it Cvt19.

Targeting of aminopeptidase I to the yeast vacuole is mediated by Ssa1p, a cytosolic member of the 70-kDa stress protein family.

The two cytosolic members of the highly conserved 70-kDa stress protein family, Ssa1p and Ssa2p, were specifically retained by the prepro-NH(2) extension of the vacuolar aminopeptidase I precursor (pAPI) conjugated to agarose (Sulfolink). A temperature-sensitive mutant strain a1(ts)a234 (ssa1(ts) ssa2 ssa3 ssa4), when incubated at the restrictive temperature, was able to assemble the API precursor into dodecamers, but failed to pack pAPI into vesicles and to convert it into mature API (mAPI), a process that occurs in the vacuole. Altogether these results indicate that Ssa1p mediates the targeting of pAPI to the vacuole.

Disruption of six novel ORFs from Saccharomyces cerevisiae chromosome VII and phenotypic analysis of the deletants.

We describe the disruption and basic phenotypic analysis of six open reading frames (ORFs) of unknown function located in the left arm of Saccharomyces cerevisiae chromosome VII, namely YGL133w, YGL134w, YGL136c, YGL138c, YGL142c and YGL144c. Disruptions were made using the short flanking homology PCR replacement strategy in the FY1679 and CEN.PK2 diploid strains. Sporulation and tetrad analysis of the heterozygous deletants was performed, as well as phenotypic analysis of the corresponding deleted haploid strains. No obvious phenotypes could be attributed to the strains deleted in any of the genes YGL134w, YGL138c and YGL144c under the conditions tested. YGL142c was shown to be an essential gene. Segregants bearing a deletion in YGL136c grew slowly in complete glycerol medium at 37 degrees C. Cells deleted in YGL133w showed abnormal morphology and reduced mating efficiency, but these phenotypes were observed only when the YGL133w disruption was in a MATalpha background. Ygl133 protein was found to localize to the nucleus.

Functional domain analysis of the yeast ABC transporter Ycf1p by site-directed mutagenesis.

The yeast cadmium factor (Ycf1p) is a vacuolar protein involved in resistance to Cd(2+) and to exogenous glutathione S-conjugate precursors in yeast. It belongs to the superfamily of ATP binding cassette transporters, which includes the human cystic fibrosis transmembrane conductance regulator and the multidrug resistance-associated protein. To examine the functional significance of conserved amino acid residues in Ycf1p, we performed an extensive mutational analysis. Twenty-two single amino acid substitutions or deletions were generated by site-directed mutagenesis in the nucleotide binding domains, the proposed regulatory domain, and the fourth cytoplasmic loop. Mutants were analyzed phenotypically by measuring their ability to grow in the presence of Cd(2+). Expression and subcellular localization of the mutant proteins were examined by immunodetection in vacuolar membranes. For functional characterization of the Ycf1p variants, the kinetic parameters of glutathione S-conjugated leukotriene C(4) transport were measured. Our analysis shows that residues Ile(711), Leu(712), Phe(713), Glu(927), and Gly(1413) are essential for Ycf1p expression. Five other amino acids, Gly(663), Gly(756), Asp(777), Gly(1306), and Gly(1311), are critical for Ycf1p function, and two residues, Glu(709) and Asp(821), are unnecessary for Ycf1p biogenesis and function. We also identify several regulatory domain mutants in which Cd(2+) tolerance of the mutant strain and transport activity of the protein are dissociated.

The prepropeptide of vacuolar aminopeptidase I is necessary and sufficient to target the fluorescent reporter protein GFP to the vacuole of yeast by the Ccvt pathway.

We have studied the capacity of the prepro amino extension of vacuolar protease leucine aminopeptidase I (API) to target the fluorescent reporter protein GFP to the vacuole of yeast. The preproGFP chimera constructed by extending the amino end of GFP with the prepro-part of API is rapidly degraded in both wild-type WCG cells and WCG 11/21a cells deficient in the proteasome. In contrast, the chimera expressed in WCG-PP cells deficient in both proteasome activity and vacuolar proteinase A accumulates in the vacuole, where it remains stable. Replacement of Gly by Ile-7, a substitution that prevents folding of the pre-part into an amphipathic helix and inhibits the targeting of the API precursor to the vacuole, inhibits the targeting of preproGFP to the vacuole. The separated pre- and pro-parts of the API precursor do not target GFP to the vacuole. Targeting of preproGFP to the vacuole is independent of its levels of expression, as the fluorescent protein localizes to the vacuole in cells expressing the protein under the control of both the GAL 1/10 or the API promoter. The preproGFP expressed under both promoters is recovered as monomers from cytosolic cell extracts. PreproGFP expressed under the API promoter is packed into cytoplasmic bodies that penetrate into the vacuolar lumen to release the protein. Altogether our results show that the prepro-part of the API precursor is necessary and sufficient to target the green fluorescent reporter protein to the vacuole.

Saccharomyces cerevisiae GPI10, the functional homologue of human PIG-B, is required for glycosylphosphatidylinositol-anchor synthesis.

An increasing number of plasma membrane proteins have been shown to be attached to the membrane via a glycosylphosphatidylinositol (GPI) moiety. All eukaryotes share a highly conserved GPI-core structure EthN-P-Man3-GlcN-PI, where EthN is ethanolamine. We have identified a protein encoded by the yeast open reading frame YGL142C that shares 33% identity with the human Pig-B protein. Deletion of this essential gene leads to a block in GPI anchor biosynthesis. We therefore named the gene GPI10. Gpi10p and Pig-B are functional homologues and the lethal deletion of GPI10 can be rescued by expression of the PIG-B cDNA. As found for PIG-B mutant cells, gpi10 deletant cells cannot attach the third mannose in an alpha-1,2 linkage to the GPI core-structure intermediate. Overexpression of GPI10 gives partial resistance to the GPI-synthesis inhibitor YW3548, suggesting that this gene product may affect the target of the inhibitor.

Sequence analysis of a 14.6 kb DNA fragment of Saccharomyces cerevisiae chromosome VII reveals SEC27, SSM1b, a putative S-adenosylmethionine-dependent enzyme and six new open reading frames.

The nucleotide sequence of a fragment from the left arm of Saccharomyces cerevisiae chromosome VII has been determined. Analysis of the 14,607 bp DNA segment reveals nine open reading frames (ORFs) longer than 300 bp. G2827 is the SEC 7 gene, an essential coatomer complex subunit. G2834 encodes SSM1b, a ribosomal protein. The G2838 product shows homology to hypothetical yeast proteins, YIF0 and YE09, of unknown function. The G2830 product shows homology with the cell division protein FtsJ from Escherichia coli, with two hypothetical proteins from yeast, YCF4 and YBR1, and with R74.7, a hypothetical protein from Caenorhabditis elegans. Two of the ORFs are completely internal to longer ones and a third is partially embedded in G2850. The remaining ORFs give no significant homology with proteins in the databases.

Transcriptional control of yeast plasma membrane H(+)-ATPase by glucose. Cloning and characterization of a new gene involved in this regulation.

The expression of the ATPase gene (PMA1) is regulated by glucose (Rao, R., Drummond-Barbosa, D., and Slayman, C. W. (1993) Yeast 9, 1075-1084) and by the TUF/RAP1/GRF1 transcription factor (Capieaux, E., Vignais, M.-L., Sentenac, A., and Goffeau, A. (1989) J. Biol. Chem. 264, 7437-7446). In this work, we describe the isolation of mutations on seven genes that affect the levels of ATPase. One of these genes (APA1) was cloned by complementation and shown to encode a protein with six putative transmembrane stretches. Expression of APA1 gene is regulated by the carbon source and requires the protein GCR1. Deletion of APA1 causes a defective regulation of the PMA1 expression by glucose but has not noticeable effect on the expression of other TUF-regulated genes. Nevertheless the expression of glucose-repressible HXT3 and SNF3 genes is significantly reduced. These results suggest a model in which APA1 acts on a glucose-signaling pathway that controls the expression of several genes that are transcriptionally regulated by glucose.

cAMP-dependent protein kinase is not involved in catabolite inactivation of the transport of sugars in Saccharomyces cerevisiae.

It has been reported that catabolite inactivation of sugar transport systems in Saccharomyces cerevisiae requires cAMP-dependent protein kinase activity (cAPK) and that the levels of these transport systems are decreased in the absence of a functional cAPK regulatory subunit. We have re-examined these possibilities and have found that catabolite inactivation does not require cAPK activity and that normal levels of the transports occur independently from the presence of the regulatory subunit. With the available information, it is difficult to ascertain the reasons for the discrepancy between our results and the ones previously reported. The inadequacy of the method used to measure the sugar transport activities might contribute to this discrepancy.

Low activity of the yeast cAMP-dependent protein kinase catalytic subunit Tpk3 is due to the poor expression of the TPK3 gene.

Three genes TPK1, TPK2 and TPK3 encode in Saccharomyces cerevisiae distinct catalytic subunits of cAMP-dependent protein kinase (cAPK). We have measured cAPK activity in vitro and, indirectly, in vivo in yeast strains carrying only one of the three TPK genes. The strain containing TPK3 as the only intact TPK gene showed nearly undetectable phosphorylating activity and no TPK3 mRNA could be detected, although the cells grow normally. Overexpression of TPK3 in a high copy vector or under the control of the inducible GAL1 promoter did not by itself result in a corresponding increase in activity but coexpression of BCY1, the gene coding for the regulatory subunit, was necessary in both cases to achieve high levels of phosphorylating activity. Moreover, BCY1 overexpression not only increased Tpk3 catalytic activity but also increased the amount of TPK3 mRNA detected in Northern blots.

Chemotactic stimulation of aggregation-stage Dictyostelium cells induces rapid changes in energy metabolism, as measured by succinic thiokinase phosphorylation.

In crude mitochondrial fractions of the cellular slime mold Dictyostelium discoideum, a 38-kDa protein can be detected in phosphorylation assays under autophosphorylation conditions in SDS polyacrylamide gels. p38 can be phosphorylated in vitro using either ATP or GTP as phosphoryl donors. After stimulation of aggregation competent cells with the chemoattractant cAMP, p38 phosphorylation pattern changes rapidly. Caffeine, a known inhibitor of cAMP relay in D. discoideum inhibits cAMP induced changes in p38 phosphorylation. The rapid changes in p38 phosphorylation after cAMP stimulation reflect changes in energy metabolism and these changes are most likely mediated by changes in internal calcium concentrations. The mitochondrial localization and other data presented on the characterization of this protein led us to the conclusion that p38 is the alpha subunit of succinic thiokinase. Data showing a correlation between in-vitro p38 phosphorylation and the metabolic state of the cells at the moment of the cell lysis are included.

cAMP- and RAS-independent nutritional regulation of plasma-membrane H+-ATPase activity in Saccharomyces cerevisiae.

The plasma-membrane ATPase of Saccharomyces cerevisiae is a proton pump whose activity, essential fro proliferation, is subject to regulation by nutritional signals. The previous finding that the CDC25 gene product is required for the glucose-induced H+-ATPase activation suggested that H+-ATPase activity is regulated by cAMP. Analysis of starvation-induced inactivation and glucose-induced activation of the H+-ATPase in mutants affected in activity of the RAS proteins, adenylyl cyclase or cAMP-dependent protein kinase showed that nutritional regulation of H+-ATPase activity does not depend directly on any of these factors. We conclude that adenlyl cyclase does not mediate all nutritional responses. This also indicates that the specific CDC25 requirement for the glucose-induced activation of the H+-ATPase identifies a new function for the CDC25 gene product, a function that appears to be independent of CDC25-mediated modulation of the RAS/adenylyl cyclase/cAMP pathway.

Yeast cAMP-dependent protein kinase can be associated to the plasma membrane.

Using an anti-yeast regulatory subunit antibody and the synthetic peptide Kemptide as specific substrate we show in this work that purified preparations of yeast plasma membrane have an associated form of the regulatory subunit and cAMP-dependent protein kinase activity. Treatment of the plasma membrane "in vitro" with 1 microM cAMP releases cAMP-independent protein kinase activity while regulatory subunit remains on the membrane as revealed by immunoblotting. Incubation of the plasma membrane with [gamma-32P]ATP results in the phosphorylation of the regulatory subunit.

Internal acidification and cAMP increase are not correlated in Saccharomyces cerevisiae.

Addition of glucose to a yeast suspension can produce both an increase in the level of cAMP and a decrease in the intracellular pH. This observation led to the idea that internal acidification triggers the cAMP increase. We have tested this hypothesis using different approaches. To study the effect of sugar metabolism on internal pH we added to the yeast either glucose or a sugar, like xylose, that cannot be phosphorylated. We also utilized yeast strains lacking hexose kinases or phosphoglucose isomerase. We found that phosphorylation of the sugar added is a requisite for internal acidification but not for the cAMP increase. Internal acidification is due to an imbalance between the rate of the metabolic reactions that generate protons and the rate at which protons can be pumped out of the cell. We have manipulated the excretion of protons by using yeast harvested at different phases of growth and resuspended in a medium with or without added K+. Addition of glucose produced a marked drop in internal pH only when the yeast was harvested in the stationary phase of growth and transferred to a medium without added K+. In contrast an increase in cAMP was observed in all situations. We conclude that in yeast there is no correlation between internal acidification and cAMP increase.

The Saccharomyces cerevisiae start mutant carrying the cdc25 mutation is defective in activation of plasma membrane ATPase by glucose.

Activation of plasma membrane ATPase by the addition of glucose was examined in several cell division cycle mutants of Saccharomyces cerevisiae. The start mutant carrying the cdc25 mutation was shown to be defective in ATPase activation at the restrictive temperature. Genetic analysis showed that lack of growth and defective activation of ATPase at the restrictive temperature were caused by the same mutation. It was also found that CDC25 does not map at the same locus as the structural gene of plasma membrane ATPase (PMA1). We conclude that the product of CDC25 controls the activation of ATPase.

Activation of yeast plasma membrane ATPase by phorbol ester.

Addition of 12-O-tetradecanoylphorbol-13-acetate (TPA) to yeast cells produces a 2-fold activation of the plasma membrane ATPase. The activation is reversible and time-and dose-dependent. The activated enzyme shows an increased affinity for its substrate, ATP, and its optimum pH is shifted to a more alkaline range. These changes are similar to those observed in the reported activation by glucose. Upon incubation of yeast cells with 32Pi incorporation of radioactivity in a membrane polypeptide of 105 kDa is observed after addition of either glucose or TPA.

Identification of phosphotyrosine in yeast proteins and of a protein tyrosine kinase associated with the plasma membrane.

[32P]Phosphotyrosine was detected in a hydrolysate of yeast proteins after in vivo labeling with [32P]phosphoric acid. The phosphoamino acid was present in cells exponentially growing on glucose as well as in cells that had reached the stationary phase of growth. Also, a plasma membrane preparation was shown to phosphorylate casein on tyrosine residues.

Fructose 2,6-bisphosphate activates the cAMP-dependent phosphorylation of yeast fructose-1,6-bisphosphatase in vitro.

Fructose-1,6-bisphosphatase purified from Saccharomyces cerevisiae is phosphorylated in vitro by a cAMP-dependent protein kinase. The phosphorylation reaction incorporates 1 mol of phosphate/mol of enzyme and is greatly stimulated by fructose 2,6-bisphosphate. Fructose 2,6-bisphosphate acts upon fructose-1,6-bisphosphatase, not on the protein kinase. The phosphorylation of fructose 1,6-bisphosphatase lowers its activity by about 50%. The characteristics of the phosphorylation reaction in vitro show that this modification is responsible for the inactivation of fructose-1,6-bisphosphatase observed in vivo.