scholarly journals Only one of the charged amino acids located in membrane-spanning regions is important for the function of the Saccharomyces cerevisiae uracil permease

1999 ◽  
Vol 339 (1) ◽  
pp. 37-42 ◽  
Author(s):  
Benoît PINSON ◽  
Jean CHEVALLIER ◽  
Danièle URBAN-GRIMAL
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Secretory Pathway ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Yeast Saccharomyces Cerevisiae ◽  
Whole Cells ◽  
Transmembrane Segments ◽  
Charged Residues ◽  
Membrane Spanning

The transport of uracil into the yeast Saccharomyces cerevisiae is mediated by uracil permease, a specific co-transporter encoded by the FUR4 gene. Uracil permease is a multispan membrane protein that is delivered to the plasma membrane via the secretory pathway. Experimental results led to the proposal of a two-dimensional model of the protein's topology. According to this model, the membrane domain of Fur4p contains three charged amino acid residues (Glu-243, Lys-272 and Glu-539) that are conserved in the members of the FUR family of yeast transporters. We have previously shown that a mis-sense mutation leading to the replacement of Lys-272 by Glu severely impairs the function of uracil permease. In the present paper, the role of the three charged residues present in the membrane-spanning regions of Fur4p was further investigated by using site-directed mutagenesis. The variant permeases were correctly targeted to the plasma membrane and their stabilities were similar to that of the wild-type permease. The effect of the mutations was studied by measuring the uptake constants for uracil on whole cells and equilibrium binding parameters on plasma membrane-enriched fractions. We found no evidence for ionic interaction between either of the glutamic residues in transmembrane segments 3 and 9 and the lysine residue in transmembrane segment 4. Of the three charged residues, only Lys-272 was important for the transport activity of the transporter. Its replacement by Ala, Glu or even Arg strongly impaired both the binding and the translocation of uracil.

scholarly journals Replacement of Lys by Glu in a transmembrane segment strongly impairs the function of the uracil permease from Saccharomyces cerevisiae

1995 ◽  
Vol 308 (3) ◽  
pp. 847-851 ◽  
Author(s):  
D Urban-Grimal ◽  
B Pinson ◽  
J Chevallier ◽  
R Haguenauer-Tsapis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Secretory Pathway ◽  
Fold Increase ◽  
Recent Experimental Data ◽  
Spontaneous Mutant ◽  
Wild Type ◽  
Transmembrane Segment ◽  
Yeast Saccharomyces Cerevisiae ◽  
Equilibrium Binding

The co-transport of uracil and protons through the plasma membrane of the yeast Saccharomyces cerevisiae is mediated by a specific permease encoded by the FUR4 gene. The uracil permease is a multi-spanning membrane protein that follows the secretory pathway to the plasma membrane. Recent experimental data led to the proposal of a two-dimensional model of its topology. A spontaneous mutant corresponding to the substitution of Lys-272 by glutamic acid was obtained. The influence of this mutation was studied by comparing the wild-type and mutant permeases produced in a strain carrying a chromosomal deletion of the FUR4 gene. The mutant permease is correctly targeted to the plasma membrane and its stability is similar to that of the wild-type permease. The uptake parameters for the mutant permease were impaired and showed an approximately 65-fold increase of apparent K(m) and a decrease in apparent Vmax. Equilibrium binding measurements with enriched plasma membrane preparations showed an approximately 70-fold increase in apparent Kd in the mutant, whereas its Bmax. was similar to that of the wild type. Lys-272 is fully conserved in the uracil permease family and is predicted to lie in the fourth transmembrane segment of the protein. It seems to be essential for both efficient uracil binding and translocation.

scholarly journals Functional Role of Charged Residues in the Transmembrane Segments of the Yeast Plasma Membrane H+-ATPase

2000 ◽  
Vol 275 (21) ◽  
pp. 15709-15716 ◽  
Author(s):  
Valery V. Petrov ◽  
Kristine P. Padmanabha ◽  
Robert K. Nakamoto ◽  
Kenneth E. Allen ◽  
Carolyn W. Slayman
Keyword(s):  
Plasma Membrane ◽  
Functional Role ◽  
Yeast Plasma Membrane ◽  
Transmembrane Segments ◽  
Charged Residues

The immunosuppressant FK506 inhibits amino acid import in Saccharomyces cerevisiae

1993 ◽  
Vol 13 (8) ◽  
pp. 5010-5019 ◽  
Author(s):  
J Heitman ◽  
A Koller ◽  
J Kunz ◽  
R Henriquez ◽  
A Schmidt ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Cyclosporin A ◽  
Secretory Pathway ◽  
Yeast Cells ◽  
Amino Acid Transporters ◽  
Yeast Saccharomyces Cerevisiae ◽  
Eukaryotic Microorganisms

The immunosuppressants cyclosporin A, FK506, and rapamycin inhibit growth of unicellular eukaryotic microorganisms and also block activation of T lymphocytes from multicellular eukaryotes. In vitro, these compounds bind and inhibit two different types of peptidyl-prolyl cis-trans isomerases. Cyclosporin A binds cyclophilins, whereas FK506 and rapamycin bind FK506-binding proteins (FKBPs). Cyclophilins and FKBPs are ubiquitous, abundant, and targeted to multiple cellular compartments, and they may fold proteins in vivo. Previously, a 12-kDa cytoplasmic FKBP was shown to be only one of at least two FK506-sensitive targets in the yeast Saccharomyces cerevisiae. We find that a second FK506-sensitive target is required for amino acid import. Amino acid-auxotrophic yeast strains (trp1 his4 leu2) are FK506 sensitive, whereas prototrophic strains (TRP1 his4 leu2, trp1 HIS4 leu2, and trp1 his4 LEU2) are FK506 resistant. Amino acids added exogenously to the growth medium mitigate FK506 toxicity. FK506 induces GCN4 expression, which is normally induced by amino acid starvation. FK506 inhibits transport of tryptophan, histidine, and leucine into yeast cells. Lastly, several genes encoding proteins involved in amino acid import or biosynthesis confer FK506 resistance. These findings demonstrate that FK506 inhibits amino acid import in yeast cells, most likely by inhibiting amino acid transporters. Amino acid transporters are integral membrane proteins which import extracellular amino acids and constitute a protein family sharing 30 to 35% identity, including eight invariant prolines. Thus, the second FK506-sensitive target in yeast cells may be a proline isomerase that plays a role in folding amino acid transporters during transit through the secretory pathway.

scholarly journals The a-factor transporter (STE6 gene product) and cell polarity in the yeast Saccharomyces cerevisiae.

1993 ◽  
Vol 120 (5) ◽  
pp. 1203-1215 ◽  
Author(s):  
K Kuchler ◽  
H G Dohlman ◽  
J Thorner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Gene Product ◽  
Cell Polarity ◽  
Polyclonal Antibodies ◽  
Apparent Molecular Weight ◽  
Subcellular Fractionation ◽  
Mating Pheromone ◽  
Yeast Saccharomyces Cerevisiae ◽  
Factor Secretion

STE6 gene product is required for secretion of the lipopeptide mating pheromone a-factor by Saccharomyces cerevisiae MATa cells. Radiolabeling and immunoprecipitation, either with specific polyclonal antibodies raised against a TrpE-Ste6 fusion protein or with mAbs that recognize c-myc epitopes in fully functional epitope-tagged Ste6 derivatives, demonstrated that Ste6 is a 145-kD phosphoprotein. Subcellular fractionation, various extraction procedures, and immunoblotting showed that Ste6 is an intrinsic plasma membrane-associated protein. The apparent molecular weight of Ste6 was unaffected by tunicamycin treatment, and the radiolabeled protein did not bind to concanavalin A, indicating that Ste6 is not glycosylated and that glycosylation is not required either for its membrane delivery or its function. The amino acid sequence of Ste6 predicts two ATP-binding folds; correspondingly, Ste6 was photoaffinity-labeled specifically with 8-azido-[alpha-32P]ATP. Indirect immunofluorescence revealed that in exponentially growing MATa cells, the majority of Ste6 showed a patchy distribution within the plasma membrane, but a significant fraction was found concentrated in a number of vesicle-like bodies subtending the plasma membrane. In contrast, in MATa cells exposed to the mating pheromone alpha-factor, which markedly induced Ste6 production, the majority of Ste6 was incorporated into the plasma membrane within the growing tip of the elongating cells. The highly localized insertion of this transporter may establish pronounced anisotropy in a-factor secretion from the MATa cell, and thereby may contribute to the establishment of the cell polarity which restricts partner selection and cell fusion during mating to one MAT alpha cell.

scholarly journals Secretory Pathway-Dependent Localization of the Saccharomyces cerevisiae Rho GTPase-Activating Protein Rgd1p at Growth Sites

2012 ◽  
Vol 11 (5) ◽  
pp. 590-600 ◽  
Author(s):  
Fabien Lefèbvre ◽  
Valérie Prouzet-Mauléon ◽  
Michel Hugues ◽  
Marc Crouzet ◽  
Aurélie Vieillemard ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Cell Cycle Progression ◽  
Secretory Pathway ◽  
Rho Gtpase ◽  
Secretory Vesicles ◽  
Gtpase Activating Protein ◽  
Content Type

ABSTRACT Establishment and maintenance of cell polarity in eukaryotes depends upon the regulation of Rho GTPases. In Saccharomyces cerevisiae , the Rho GTPase activating protein (RhoGAP) Rgd1p stimulates the GTPase activities of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively. Consistent with the distribution of Rho3p and Rho4p, Rgd1p is found mostly in areas of polarized growth during cell cycle progression. Rgd1p was mislocalized in mutants specifically altered for Golgi apparatus-based phosphatidylinositol 4-P [PtdIns(4)P] synthesis and for PtdIns(4,5)P 2 production at the plasma membrane. Analysis of Rgd1p distribution in different membrane-trafficking mutants suggested that Rgd1p was delivered to growth sites via the secretory pathway. Rgd1p may associate with post-Golgi vesicles by binding to PtdIns(4)P and then be transported by secretory vesicles to the plasma membrane. In agreement, we show that Rgd1p coimmunoprecipitated and localized with markers specific to secretory vesicles and cofractionated with a plasma membrane marker. Moreover, in vivo imaging revealed that Rgd1p was transported in an anterograde manner from the mother cell to the daughter cell in a vectoral manner. Our data indicate that secretory vesicles are involved in the delivery of RhoGAP Rgd1p to the bud tip and bud neck.

scholarly journals The Methionine 549 and Leucine 552 Residues of Friedelin Synthase from Maytenus ilicifolia Are Important for Substrate Binding Specificity

Molecules ◽  
2021 ◽  
Vol 26 (22) ◽  
pp. 6806
Author(s):  
Bruna F. Mazzeu ◽  
Tatiana M. Souza-Moreira ◽  
Andrew A. Oliveira ◽  
Melissa Remlinger ◽  
Lidiane G. Felippe ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Biological Activities ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Enzyme Specificity ◽  
Amino Acid Residues ◽  
Loss Of Function ◽  
Pentacyclic Triterpene ◽  
Oxidosqualene Cyclases ◽  
Maytenus Ilicifolia

Friedelin, a pentacyclic triterpene found in the leaves of the Celastraceae species, demonstrates numerous biological activities and is a precursor of quinonemethide triterpenes, which are promising antitumoral agents. Friedelin is biosynthesized from the cyclization of 2,3-oxidosqualene, involving a series of rearrangements to form a ketone by deprotonation of the hydroxylated intermediate, without the aid of an oxidoreductase enzyme. Mutagenesis studies among oxidosqualene cyclases (OSCs) have demonstrated the influence of amino acid residues on rearrangements during substrate cyclization: loss of catalytic activity, stabilization, rearrangement control or specificity changing. In the present study, friedelin synthase from Maytenus ilicifolia (Celastraceae) was expressed heterologously in Saccharomyces cerevisiae. Site-directed mutagenesis studies were performed by replacing phenylalanine with tryptophan at position 473 (Phe473Trp), methionine with serine at position 549 (Met549Ser) and leucine with phenylalanine at position 552 (Leu552Phe). Mutation Phe473Trp led to a total loss of function; mutants Met549Ser and Leu552Phe interfered with the enzyme specificity leading to enhanced friedelin production, in addition to α-amyrin and β-amyrin. Hence, these data showed that methionine 549 and leucine 552 are important residues for the function of this synthase.

scholarly journals Identification of Amino Acid Residues in the α, β, and γ Subunits of the Epithelial Sodium Channel (ENaC) Involved in Amiloride Block and Ion Permeation

1997 ◽  
Vol 109 (1) ◽  
pp. 15-26 ◽  
Author(s):  
Laurent Schild ◽  
Estelle Schneeberger ◽  
Ivan Gautschi ◽  
Dmitri Firsov
Keyword(s):  
Amino Acid ◽  
Ion Selectivity ◽  
Site Directed Mutagenesis ◽  
Ion Permeation ◽  
Amino Acid Residues ◽  
Α Subunit ◽  
Na Ions ◽  
Membrane Spanning ◽  
A Site ◽  
Epithelial Sodium Channel Enac

The amiloride-sensitive epithelial Nachannel (ENaC) is a heteromultimeric channel made of three αβγ subunits. The structures involved in the ion permeation pathway have only been partially identified, and the respective contributions of each subunit in the formation of the conduction pore has not yet been established. Using a site-directed mutagenesis approach, we have identified in a short segment preceding the second membrane-spanning domain (the pre-M2 segment) amino acid residues involved in ion permeation and critical for channel block by amiloride. Cys substitutions of Gly residues in β and γ subunits at position βG525 and γG537 increased the apparent inhibitory constant (Ki) for amiloride by >1,000-fold and decreased channel unitary current without affecting ion selectivity. The corresponding mutation S583 to C in the α subunit increased amiloride Ki by 20-fold, without changing channel conducting properties. Coexpression of these mutated αβγ subunits resulted in a nonconducting channel expressed at the cell surface. Finally, these Cys substitutions increased channel affinity for block by externalZn2+ ions, in particular the αS583C mutant showing a Ki for Zn2+of 29 μM. Mutations of residues αW582L or βG522D also increased amiloride Ki, the later mutation generating a Ca2+blocking site located 15% within the membrane electric field. These experiments provide strong evidence that αβγ ENaCs are pore-forming subunits involved in ion permeation through the channel. The pre-M2 segment of αβγ subunits may form a pore loop structure at the extracellular face of the channel, where amiloride binds within the channel lumen. We propose that amiloride interacts with Na+ions at an external Na+binding site preventing ion permeation through the channel pore.

Identification of pre-mRNA polyadenylation sites in Saccharomyces cerevisiae

1992 ◽  
Vol 12 (9) ◽  
pp. 4215-4229
Author(s):  
S Heidmann ◽  
B Obermaier ◽  
K Vogel ◽  
H Domdey
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Signal Sequence ◽  
Site Directed Mutagenesis ◽  
Yeast Cells ◽  
Yeast Saccharomyces Cerevisiae ◽  
Adh1 Gene ◽  
Polyadenylation Sites ◽  
Yeast Genes

In contrast to higher eukaryotes, little is known about the nature of the sequences which direct 3'-end formation of pre-mRNAs in the yeast Saccharomyces cerevisiae. The hexanucleotide AAUAAA, which is highly conserved and crucial in mammals, does not seem to have any functional importance for 3'-end formation in yeast cells. Instead, other elements have been proposed to serve as signal sequences. We performed a detailed investigation of the yeast ACT1, ADH1, CYC1, and YPT1 cDNAs, which showed that the polyadenylation sites used in vivo can be scattered over a region spanning up to 200 nucleotides. It therefore seems very unlikely that a single signal sequence is responsible for the selection of all these polyadenylation sites. Our study also showed that in the large majority of mRNAs, polyadenylation starts directly before or after an adenosine residue and that 3'-end formation of ADH1 transcripts occurs preferentially at the sequence PyAAA. Site-directed mutagenesis of these sites in the ADH1 gene suggested that this PyAAA sequence is essential for polyadenylation site selection both in vitro and in vivo. Furthermore, the 3'-terminal regions of the yeast genes investigated here are characterized by their capacity to act as signals for 3'-end formation in vivo in either orientation.

Signals that produce 3' termini in CYC1 mRNA of the yeast Saccharomyces cerevisiae

1993 ◽  
Vol 13 (12) ◽  
pp. 7836-7849
Author(s):  
P Russo ◽  
W Z Li ◽  
Z Guo ◽  
F Sherman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Site Directed Mutagenesis ◽  
Yeast Cells ◽  
In Vitro Mutagenesis ◽  
Yeast Saccharomyces Cerevisiae ◽  
Upstream Element ◽  
A Site ◽  
Derivatives Of ◽  
Acting In Concert

The cyc1-512 mutant was previously shown to contain a 38-bp deletion, 8 nucleotides upstream from the major wild-type poly(A) site, in the CYC1 gene, which encodes iso-1-cytochrome c of the yeast Saccharomyces cerevisiae. This 38-bp deletion caused a 90% reduction in the CYC1 transcripts, which were heterogeneous in size, aberrantly long, and presumably labile (K. S. Zaret and F. Sherman, Cell 28:563-573, 1982). Site-directed mutagenesis in and adjacent to the 38-bp region was used to identify signals involved in the formation and positioning of CYC1 mRNA 3' ends. In addition, combinations of various putative 3' end-forming signals were introduced by in vitro mutagenesis into the 3' region of the cyc1-512 mutant. The combined results from both studies suggest that 3' end formation in yeast cells involves signals having the following three distinct but integrated elements acting in concert: (i) the upstream element, including sequences TATATA, TAG ... TATGTA, and TTTTTATA, which function by enhancing the efficiency of downstream elements; (ii) downstream elements, such as TTAAGAAC and AAGAA, which position the poly(A) site; and (iii) the actual site of polyadenylation, which often occurs after cytidine residues that are 3' to the so-called downstream element. While the upstream element is required for efficient 3' end formation, alterations of the downstream element and poly(A) sites generally do not affect the efficiency of 3' end formation but appear to alter the positions of poly(A) sites. In addition, we have better defined the upstream elements by examining various derivatives of TATATA and TAG ... TATGTA, and we have examined the spatial requirements of the three elements by systematically introducing or deleting upstream and downstream elements and cytidine poly(A) sites.

scholarly journals Amino acid residues in the CDC25 guanine nucleotide exchange factor critical for interaction with Ras.

1994 ◽  
Vol 14 (12) ◽  
pp. 8117-8122 ◽  
Author(s):  
W Park ◽  
R D Mosteller ◽  
D Broek
Keyword(s):  
Guanine Nucleotide Exchange Factor ◽  
Ion Pair ◽  
Site Directed Mutagenesis ◽  
Guanine Nucleotide ◽  
Amino Acid Residues ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Charged Residues

Previously we found that negatively charged residues at positions 62, 63, and 69 of H-Ras are involved in binding to the CDC25 guanine nucleotide exchange factor (GEF). Using site-directed mutagenesis, we have changed conserved, positively charged residues of CDC25GEF to glutamic acid. We find the nonfunctional CDC25R1374E mutant and the nonfunctional H-RasE63K mutant cooperate in suppression of the loss of CDC25 function in Saccharomyces cerevisiae. Also, peptides corresponding to residues 1364 to 1383 of CDC25GEF inhibit interaction between GEFs and H-Ras. We propose that residues 1374 of CDC25GEF and 63 of H-Ras form an ion pair and that when this ion pair is reversed, functional interaction can still occur.

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