scholarly journals Complex Formation with Ypt11p, a rab-Type Small GTPase, Is Essential To Facilitate the Function of Myo2p, a Class V Myosin, in Mitochondrial Distribution in Saccharomyces cerevisiae

2002 ◽  
Vol 22 (22) ◽  
pp. 7744-7757 ◽  
Author(s):  
Takashi Itoh ◽  
Akiko Watabe ◽  
Akio Toh-e ◽  
Yasushi Matsui
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Complex Formation ◽  
Mutational Analysis ◽  
Small Gtpase ◽  
Tail Domain ◽  
Class V ◽  
Synthetic Lethal ◽  
Partial Delay ◽  
Mitochondrial Distribution

ABSTRACT We identified Ypt11p, a rab-type small GTPase, by its functional and two-hybrid interaction with Myo2p, a class V myosin of the budding yeast Saccharomyces cerevisiae. The tail domain of Myo2p was coimmunoprecipitated with Ypt11p, suggesting that Ypt11p forms a complex with Myo2p at its tail domain in vivo. Mutational analysis of YPT11 suggests that Myo2p is a putative effector of Ypt11p. Deletion of YPT11 induced partial delay of mitochondrial transmission to the bud, and overexpression of YPT11 resulted in mitochondrial accumulation in the bud, indicating that Ypt11p acts positively on mitochondrial distribution toward the bud. We isolated two myo2 mutants, myo2-338 and myo2-573, which showed genetic interactions with YPT11. The myo2-573 mutation, identified by a synthetic lethal interaction with ypt11-null, induced a defect in mitochondrial distribution toward the bud, indicating that Myo2p plays a crucial role in polarized distribution of mitochondria. The myo2-338 mutation was identified as the mutation that abolished the effect of overexpressed YPT11, such as the Ypt11p-dependent accumulation of mitochondria in the bud, and the affinity of Myo2p for Ypt11p was reduced. These results indicate that complex formation of Ypt11p with Myo2p accelerates the function of Myo2p for mitochondrial distribution toward the bud.

scholarly journals Mutational analysis of the iron binding site of Saccharomyces cerevisiae ferroxidase Fet3. An in vivo study

FEBS Letters ◽  
2001 ◽  
Vol 508 (3) ◽  
pp. 475-478 ◽  
Author(s):  
Maria Carmela Bonaccorsi di Patti ◽  
Maria Paola Paronetto ◽  
Valeria Dolci ◽  
Maria Rosa Felice ◽  
Amalia Lania ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Site ◽  
Mutational Analysis ◽  
In Vivo Study ◽  
Iron Binding

The Saccharomyces cerevisiae PUT3 activator protein associates with proline-specific upstream activation sequences

1989 ◽  
Vol 9 (11) ◽  
pp. 4706-4712
Author(s):  
A H Siddiqui ◽  
M C Brandriss
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Complex Formation ◽  
Binding Activity ◽  
Lacz Gene ◽  
Wild Type ◽  
Mobility Shift ◽  
Proline Utilization

The PUT1 and PUT2 genes encoding the enzymes of the proline utilization pathway of Saccharomyces cerevisiae are induced by proline and activated by the product of the PUT3 gene. Two upstream activation sequences (UASs) in the PUT1 promoter were identified by homology to the PUT2 UAS. Deletion analysis of the two PUT1 UASs showed that they were functionally independent and additive in producing maximal levels of gene expression. The consensus PUT UAS is a 21-base-pair partially palindromic sequence required in vivo for induction of both genes. The results of a gel mobility shift assay demonstrated that the proline-specific UAS is the binding site of a protein factor. In vitro complex formation was observed in crude extracts of yeast strains carrying either a single genomic copy of the PUT3 gene or the cloned PUT3 gene on a 2 microns plasmid, and the binding was dosage dependent. DNA-binding activity was not observed in extracts of strains carrying either a put3 mutation that caused a noninducible (Put-) phenotype or a deletion of the gene. Wild-type levels of complex formation were observed in an extract of a strain carrying an allele of PUT3 that resulted in a constitutive (Put+) phenotype. Extracts from a strain carrying a PUT3-lacZ gene fusion formed two complexes of slower mobility than the wild-type complex. We conclude that the PUT3 product is either a DNA-binding protein or part of a DNA-binding complex that recognizes the UASs of both PUT1 and PUT2. Binding was observed in extracts of a strain grown in the presence or absence of proline, demonstrating the constitutive nature of the DNA-protein interaction.

In Vivo Mutational Analysis of Ribosomal RNA in Saccharomyces cerevisiae

2003 ◽  
pp. 257-270 ◽  
Author(s):  
Jaap Venema ◽  
Rudi J. Planta ◽  
Hendrik A. Raué
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Rna ◽  
Mutational Analysis

scholarly journals Mutational analysis of conserved positions potentially important for initiator tRNA function in Saccharomyces cerevisiae.

1992 ◽  
Vol 12 (4) ◽  
pp. 1432-1442 ◽  
Author(s):  
U von Pawel-Rammingen ◽  
S Aström ◽  
A S Byström
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Growth ◽  
Base Pair ◽  
Mutational Analysis ◽  
Null Alleles ◽  
Nucleotide Substitutions ◽  
Double Mutation ◽  
Initiator Trna ◽  
Initiation Of Translation

The conserved positions of the eukaryotic cytoplasmic initiator tRNA have been suggested to be important for the initiation of protein synthesis. However, the role of these positions is not known. We describe in this report a functional analysis of the yeast initiator methionine tRNA (tRNA(iMet)), using a novel in vivo assay system which is not dependent on suppressor tRNAs. Strains of Saccharomyces cerevisiae with null alleles of the four initiator methionine tRNA (IMT) genes were constructed. Consequently, growth of these strains was dependent on tRNA(iMet) encoded from a plasmid-derived gene. We used these strains to investigate the significance of the conserved nucleosides of yeast tRNA(iMet) in vivo. Nucleotide substitutions corresponding to the nucleosides of the yeast elongator methionine tRNA (tRNA(MMet)) have been made at all conserved positions to identify the positions that are important for tRNA(iMet) to function in the initiation process. Surprisingly, nucleoside changes in base pairs 3-70, 12-23, 31-39, and 29-41, as well as expanding loop I by inserting an A at position 17 (A17) had no effect on the tester strain. Nucleotide substitutions in positions 54 and 60 to cytidines and guanosines (C54, G54, C60, and G60) did not prevent cell growth. In contrast, the double mutation U/rT54C60 blocked cell growth, and changing the A-U base pair 1-72 to a G-C base pair was deleterious to the cell, although these tRNAs were synthesized and accepted methionine in vitro. From our data, we suggest that an A-U base pair in position 1-72 is important for tRNA(iMet) function, that the hypothetical requirement for adenosines at positions 54 and 60 is invalid, and that a U/rT at position 54 is an antideterminant distinguishing an elongator from an initiator tRNA in the initiation of translation.

scholarly journals End4p/Sla2p Interacts with Actin-associated Proteins for Endocytosis in Saccharomyces cerevisiae

1997 ◽  
Vol 8 (11) ◽  
pp. 2291-2306 ◽  
Author(s):  
A. Wesp ◽  
L. Hicke ◽  
J. Palecek ◽  
R. Lombardi ◽  
T. Aust ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
High Temperature ◽  
Mutational Analysis ◽  
Coiled Coil ◽  
Temperature Sensitive ◽  
Cortical Actin ◽  
Actin Organization ◽  
Coiled Coil Domain ◽  
Associated Proteins

end4–1 was isolated as a temperature-sensitive endocytosis mutant. We cloned and sequenced END4 and found that it is identical to SLA2/MOP2. This gene is required for growth at high temperature, viability in the absence of Abp1p, polarization of the cortical actin cytoskeleton, and endocytosis. We used a mutational analysis of END4 to correlate in vivo functions with regions of End4p and we found that two regions of End4p participate in endocytosis but that the talin-like domain of End4p is dispensable. The N-terminal domain of End4p is required for growth at high temperature, endocytosis, and actin organization. A central coiled-coil domain of End4p is necessary for formation of a soluble sedimentable complex. Furthermore, this domain has an endocytic function that is redundant with the function(s) ofABP1 and SRV2. The endocytic function of Abp1p depends on its SH3 domain. In addition we have isolated a recessive negative allele of SRV2 that is defective for endocytosis. Combined biochemical, functional, and genetic analysis lead us to propose that End4p may mediate endocytosis through interaction with other actin-associated proteins, perhaps Rvs167p, a protein essential for endocytosis.

scholarly journals Nucleosome depletion alters the chromatin structure of Saccharomyces cerevisiae centromeres.

1990 ◽  
Vol 10 (11) ◽  
pp. 5721-5727 ◽  
Author(s):  
M J Saunders ◽  
E Yeh ◽  
M Grunstein ◽  
K Bloom
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Segregation ◽  
Mutational Analysis ◽  
Base Pairs ◽  
Centromere Function ◽  
Nuclease Digestion ◽  
Dna Elements ◽  
Chromosome Iii ◽  
Sequence Requirements

Saccharomyces cerevisiae centromeric DNA is packaged into a highly nuclease-resistant chromatin core of approximately 200 base pairs of DNA. The structure of the centromere in chromosome III is somewhat larger than a 160-base-pair nucleosomal core and encompasses the conserved centromere DNA elements (CDE I, II, and III). Extensive mutational analysis has revealed the sequence requirements for centromere function. Mutations affecting the segregation properties of centromeres also exhibit altered chromatin structures in vivo. Thus the structure, as delineated by nuclease digestion, correlated with functional centromeres. We have determined the contribution of histone proteins to this unique structural organization. Nucleosome depletion by repression of either histone H2B or H4 rendered the cell incapable of chromosome segregation. Histone repression resulted in increased nuclease sensitivity of centromere DNA, with up to 40% of CEN3 DNA molecules becoming accessible to nucleolytic attack. Nucleosome depletion also resulted in an alteration in the distribution of nuclease cutting sites in the DNA surrounding CEN3. These data provide the first indication that authentic nucleosomal subunits flank the centromere and suggest that nucleosomes may be the central core of the centromere itself.

scholarly journals Nucleosome depletion alters the chromatin structure of Saccharomyces cerevisiae centromeres

1990 ◽  
Vol 10 (11) ◽  
pp. 5721-5727
Author(s):  
M J Saunders ◽  
E Yeh ◽  
M Grunstein ◽  
K Bloom
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Segregation ◽  
Mutational Analysis ◽  
Base Pairs ◽  
Centromere Function ◽  
Nuclease Digestion ◽  
Dna Elements ◽  
Chromosome Iii ◽  
Sequence Requirements

Saccharomyces cerevisiae centromeric DNA is packaged into a highly nuclease-resistant chromatin core of approximately 200 base pairs of DNA. The structure of the centromere in chromosome III is somewhat larger than a 160-base-pair nucleosomal core and encompasses the conserved centromere DNA elements (CDE I, II, and III). Extensive mutational analysis has revealed the sequence requirements for centromere function. Mutations affecting the segregation properties of centromeres also exhibit altered chromatin structures in vivo. Thus the structure, as delineated by nuclease digestion, correlated with functional centromeres. We have determined the contribution of histone proteins to this unique structural organization. Nucleosome depletion by repression of either histone H2B or H4 rendered the cell incapable of chromosome segregation. Histone repression resulted in increased nuclease sensitivity of centromere DNA, with up to 40% of CEN3 DNA molecules becoming accessible to nucleolytic attack. Nucleosome depletion also resulted in an alteration in the distribution of nuclease cutting sites in the DNA surrounding CEN3. These data provide the first indication that authentic nucleosomal subunits flank the centromere and suggest that nucleosomes may be the central core of the centromere itself.

scholarly journals The Anatomy of a Hypoxic Operator in Saccharomyces cerevisiae

Genetics ◽  
1998 ◽  
Vol 150 (4) ◽  
pp. 1429-1441 ◽  
Author(s):  
Jutta Deckert ◽  
Ana Maria Rodriguez Torres ◽  
Soo Myung Hwang ◽  
Alexander J Kastaniotis ◽  
Richard S Zitomer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Sites ◽  
Mutational Analysis ◽  
Consensus Sequence ◽  
Number Of Genes ◽  
Repression Complex ◽  
Single Base Pair ◽  
Sequence Requirements

Abstract Aerobic repression of the hypoxic genes of Saccharomyces cerevisiae is mediated by the DNA-binding protein Rox1 and the Tup1/Ssn6 general repression complex. To determine the DNA sequence requirements for repression, we carried out a mutational analysis of the consensus Rox1-binding site and an analysis of the arrangement of the Rox1 sites into operators in the hypoxic ANB1 gene. We found that single base pair substitutions in the consensus sequence resulted in lower affinities for Rox1, and the decreased affinity of Rox1 for mutant sites correlated with the ability of these sites to repress expression of the hypoxic ANB1 gene. In addition, there was a general but not complete correlation between the strength of repression of a given hypoxic gene and the compliance of the Rox1 sites in that gene to the consensus sequence. An analysis of the ANB1 operators revealed that the two Rox1 sites within an operator acted synergistically in vivo, but that Rox1 did not bind cooperatively in vitro, suggesting the presence of a higher order repression complex in the cell. In addition, the spacing or helical phasing of the Rox1 sites was not important in repression. The differential repression by the two operators of the ANB1 gene was found to be due partly to the location of the operators and partly to the sequences between the two Rox1-binding sites in each. Finally, while Rox1 repression requires the Tup1/Ssn6 general repression complex and this complex has been proposed to require the aminoterminal regions of histones H3 and H4 for full repression of a number of genes, we found that these regions were dispensable for ANB1 repression and the repression of two other hypoxic genes.

Mutational analysis of conserved positions potentially important for initiator tRNA function in Saccharomyces cerevisiae

1992 ◽  
Vol 12 (4) ◽  
pp. 1432-1442
Author(s):  
U von Pawel-Rammingen ◽  
S Aström ◽  
A S Byström
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Growth ◽  
Base Pair ◽  
Mutational Analysis ◽  
Null Alleles ◽  
Nucleotide Substitutions ◽  
Double Mutation ◽  
Initiator Trna ◽  
Initiation Of Translation

The conserved positions of the eukaryotic cytoplasmic initiator tRNA have been suggested to be important for the initiation of protein synthesis. However, the role of these positions is not known. We describe in this report a functional analysis of the yeast initiator methionine tRNA (tRNA(iMet)), using a novel in vivo assay system which is not dependent on suppressor tRNAs. Strains of Saccharomyces cerevisiae with null alleles of the four initiator methionine tRNA (IMT) genes were constructed. Consequently, growth of these strains was dependent on tRNA(iMet) encoded from a plasmid-derived gene. We used these strains to investigate the significance of the conserved nucleosides of yeast tRNA(iMet) in vivo. Nucleotide substitutions corresponding to the nucleosides of the yeast elongator methionine tRNA (tRNA(MMet)) have been made at all conserved positions to identify the positions that are important for tRNA(iMet) to function in the initiation process. Surprisingly, nucleoside changes in base pairs 3-70, 12-23, 31-39, and 29-41, as well as expanding loop I by inserting an A at position 17 (A17) had no effect on the tester strain. Nucleotide substitutions in positions 54 and 60 to cytidines and guanosines (C54, G54, C60, and G60) did not prevent cell growth. In contrast, the double mutation U/rT54C60 blocked cell growth, and changing the A-U base pair 1-72 to a G-C base pair was deleterious to the cell, although these tRNAs were synthesized and accepted methionine in vitro. From our data, we suggest that an A-U base pair in position 1-72 is important for tRNA(iMet) function, that the hypothetical requirement for adenosines at positions 54 and 60 is invalid, and that a U/rT at position 54 is an antideterminant distinguishing an elongator from an initiator tRNA in the initiation of translation.

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