Amino-terminal protein processing in Saccharomyces cerevisiae is an essential function that requires two distinct methionine aminopeptidases.

Abstract The vacuolar hydrolase protease B in Saccharomyces cerevisiae is synthesized as an inactive precursor (Prb1p). The precursor undergoes post-translational modifications while transiting the secretory pathway. In addition to N- and O -linked glycosylations, four proteolytic cleavages occur during the maturation of Prb1p. Removal of the signal peptide by signal peptidase and the autocatalytic cleavage of the large aminoterminal propeptide occur in the endoplasmic reticulum (ER). Two carboxy-terminal cleavages of the post regions occur in the vacuole: the first cleavage is catalyzed by protease A and the second results from autocatalysis. We have isolated a mutant, pbn1-1, that exhibits a defect in the ER processing of Prb1p. The autocatalytic cleavage of the propeptide from Prb1p does not occur and Prb1p is rapidly degraded in the cytosol. PBN1 was cloned and is identical to YCL052c on chromosome III. PBN1 is an essential gene that encodes a novel protein. Pbn1p is predicted to contain a sub-C-terminal transmembrane domain but no signal sequence. A functional HA epitope-tagged Pbn1p fusion localizes to the ER. Pbn1p is N-glycosylated in its amino-terminal domain, indicating a lumenal orientation despite the lack of a signal sequence. Based on these results, we propose that one of the functions of Pbn1p is to aid in the autocatalytic processing of Prb1p.

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SEN1, a positive effector of tRNA-splicing endonuclease in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.12.5.2154 ◽

1992 ◽

Vol 12 (5) ◽

pp. 2154-2164 ◽

Cited By ~ 50

Author(s):

D J DeMarini ◽

M Winey ◽

D Ursic ◽

F Webb ◽

M R Culbertson

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acids ◽

Amino Acid ◽

Gene Product ◽

Signal Sequence ◽

Null Alleles ◽

Nucleoside Triphosphate ◽

Temperature Sensitive ◽

Yeast Saccharomyces Cerevisiae ◽

Amino Terminal

The SEN1 gene, which is essential for growth in the yeast Saccharomyces cerevisiae, is required for endonucleolytic cleavage of introns from all 10 families of precursor tRNAs. A mutation in SEN1 conferring temperature-sensitive lethality also causes in vivo accumulation of pre-tRNAs and a deficiency of in vitro endonuclease activity. Biochemical evidence suggests that the gene product may be one of several components of a nuclear-localized splicing complex. We have cloned the SEN1 gene and characterized the SEN1 mRNA, the SEN1 gene product, the temperature-sensitive sen1-1 mutation, and three SEN1 null alleles. The SEN1 gene corresponds to a 6,336-bp open reading frame coding for a 2,112-amino-acid protein (molecular mass, 239 kDa). Using antisera directed against the C-terminal end of SEN1, we detect a protein corresponding to the predicted molecular weight of SEN1. The SEN1 protein contains a leucine zipper motif, consensus elements for nucleoside triphosphate binding, and a potential nuclear localization signal sequence. The carboxy-terminal 1,214 amino acids of the SEN1 protein are essential for growth, whereas the amino-terminal 898 amino acids are dispensable. A sequence of approximately 500 amino acids located in the essential region of SEN1 has significant similarity to the yeast UPF1 gene product, which is involved in mRNA turnover, and the mouse Mov-10 gene product, whose function is unknown. The mutation that creates the temperature-sensitive sen1-1 allele is located within this 500-amino-acid region, and it causes a substitution for an amino acid that is conserved in all three proteins.

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RAD3 gene of Saccharomyces cerevisiae: nucleotide sequence of wild-type and mutant alleles, transcript mapping, and aspects of gene regulation

Molecular and Cellular Biology ◽

10.1128/mcb.5.1.17-26.1985 ◽

1985 ◽

Vol 5 (1) ◽

pp. 17-26

Author(s):

L Naumovski ◽

G Chu ◽

P Berg ◽

E C Friedberg

Keyword(s):

Saccharomyces Cerevisiae ◽

Nucleotide Sequence ◽

Coding Region ◽

Base Pairs ◽

S1 Nuclease ◽

Calculated Molecular Weight ◽

Base Pair Deletion ◽

S1 Nuclease Mapping ◽

Essential Function ◽

Nuclease Mapping

We determined the complete nucleotide sequence of the RAD3 gene of Saccharomyces cerevisiae. The coding region of the gene contained 2,334 base pairs that could encode a protein with a calculated molecular weight of 89,796. Analysis of RAD3 mRNA by Northern blots and by S1 nuclease mapping indicated that the transcript was approximately 2.5 kilobases and did not contain intervening sequences. Fusions between the RAD3 gene and the lac'Z gene of Escherichia coli were constructed and used to demonstrate that the RAD3 gene was not inducible by DNA damage caused by UV radiation or 4-nitroquinoline-1-oxide. Two UV-sensitive chromosomal mutant alleles of RAD3, rad3-1 and rad3-2, were rescued by gap repair of a centromeric plasmid, and their sequences were determined. The rad3-1 mutation changed a glutamic acid to lysine, and the rad3-2 mutation changed a glycine to arginine. Previous studies have shown that disruption of the RAD3 gene results in loss of an essential function and is associated with inviability of haploid cells. In the present experiments, plasmids carrying the rad3-1 and rad3-2 mutations were introduced into haploid cells containing a disrupted RAD3 gene. These plasmids expressed the essential function of RAD3 but not its DNA repair function. A 74-base-pair deletion at the 3' end of the RAD3 coding region or a fusion of this deletion to the E. coli lac'Z gene did not affect either function of RAD3.

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Functional interaction between p21rap1A and components of the budding pathway in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.12.9.4084-4092.1992 ◽

1992 ◽

Vol 12 (9) ◽

pp. 4084-4092

Author(s):

P C McCabe ◽

H Haubruck ◽

P Polakis ◽

F McCormick ◽

M A Innis

Keyword(s):

Saccharomyces Cerevisiae ◽

Mammalian Cells ◽

Bound States ◽

Yeast Cells ◽

Temperature Sensitive ◽

Wild Type ◽

Amino Terminal ◽

Loop Region

The rap1A gene encodes a 21-kDa, ras-related GTP-binding protein (p21rap1A) of unknown function. A close structural homolog of p21rap1A (65% identity in the amino-terminal two-thirds) is the RSR1 gene product (Rsr1p) of Saccharomyces cerevisiae. Although Rsr1p is not essential for growth, its presence is required for nonrandom selection of bud sites. To assess the similarity of these proteins at the functional level, wild-type and mutant forms of p21rap1A were tested for complementation of activities known to be fulfilled by Rsr1p. Expression of p21rap1A, like multicopy expression of RSR1, suppressed the conditional lethality of a temperature-sensitive cdc24 mutation. Point mutations predicted to affect the localization of p21rap1A or its ability to cycle between GDP and GTP-bound states disrupted suppression of cdc24ts, while other mutations in the 61-65 loop region improved suppression. Expression of p21rap1A could not, however, suppress the random budding phenotype of rsr1 cells. p21rap1A also apparently interfered with the normal activity of Rsrlp, causing random budding in diploid wild-type cells, suggesting an inability of p21rap1A to interact appropriately with Rsr1p regulatory proteins. Consistent with this hypothesis, we found an Rsr1p-specific GTPase-activating protein (GAP) activity in yeast membranes which was not active toward p21rap1A, indicating that p21rap1A may be predominantly GTP bound in yeast cells. Coexpression of human Rap1-specific GAP suppressed the random budding due to expression of p21rap1A or its derivatives, including Rap1AVal-12. Although Rap1-specific GAP stimulated the GTPase of Rsr1p in vitro, it did not dominantly interfere with Rsr1p function in vivo. A chimera consisting of Rap1A1-165::Rsr1p166-272 did not exhibit normal Rsr1p function in the budding pathway. These results indicated that p21rap1A and Rsr1p share at least partial functional homology, which may have implications for p21rap1A function in mammalian cells.

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Interaction with Tap42 Is Required for the Essential Function of Sit4 and Type 2A Phosphatases

Molecular Biology of the Cell ◽

10.1091/mbc.e03-02-0072 ◽

2003 ◽

Vol 14 (11) ◽

pp. 4342-4351 ◽

Cited By ~ 61

Author(s):

Huamin Wang ◽

Xiaodong Wang ◽

Yu Jiang

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acid ◽

Amino Acid Substitution ◽

Binding Domain ◽

Charge Amino Acid ◽

Catalytic Subunits ◽

Regulatory Subunits ◽

Terminal Domain ◽

Major Form ◽

Essential Function

In Saccharomyces cerevisiae, Pph21 and Pph22 are the two catalytic subunits of type 2A phosphatase (PP2Ac), and Sit4 is a major form of 2A-like phosphatase. The function of these phosphatases requires their association with different regulatory subunits. In addition to the conventional regulatory subunits, namely, the A and B subunits for Pph21/22 and the Sap proteins for Sit4, these phosphatases have been found to associate with a protein termed Tap42. In this study, we demonstrated that Sit4 and PP2Ac interact with Tap42 via an N-terminal domain that is conserved in all type 2A and 2A-like phosphatases. We found that the Sit4 phosphatase in the sit4-102 strain contains a reverse-of-charge amino acid substitution within its Tap42 binding domain and is defective for formation of the Tap42-Sit4 complex. Our results suggest that the interaction with Tap42 is required for the activity as well as for the essential function of Sit4 and PP2Ac. In addition, we showed that Tap42 is able to interact with two other 2A-like phosphatases, Pph3 and Ppg1.

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GlyGly-CTERM and Rhombosortase: A C-Terminal Protein Processing Signal in a Many-to-One Pairing with a Rhomboid Family Intramembrane Serine Protease

PLoS ONE ◽

10.1371/journal.pone.0028886 ◽

2011 ◽

Vol 6 (12) ◽

pp. e28886 ◽

Cited By ~ 16

Author(s):

Daniel H. Haft ◽

Neha Varghese

Keyword(s):

Serine Protease ◽

Protein Processing ◽

Terminal Protein ◽

Processing Signal

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Signal peptide specificity in posttranslational processing of the plant protein phaseolin in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.7.1.121 ◽

1987 ◽

Vol 7 (1) ◽

pp. 121-128 ◽

Cited By ~ 15

Author(s):

J H Cramer ◽

K Lea ◽

M D Schaber ◽

R A Kramer

Keyword(s):

Saccharomyces Cerevisiae ◽

Acid Phosphatase ◽

Signal Peptide ◽

Regulatory Region ◽

Cellular Protein ◽

Proteolytic Processing ◽

Coding Region ◽

Molecular Weights ◽

Amino Terminal ◽

Acid Phosphatase Gene

We linked the cDNA coding region for the bean storage protein phaseolin to the promoter and regulatory region of the Saccharomyces cerevisiae repressible acid phosphatase gene (PHO5) in multicopy expression plasmids. Yeast transformants containing these plasmids expressed phaseolin at levels up to 3% of the total soluble cellular protein. Phaseolin polypeptides in S. cerevisiae were glycosylated, and their molecular weights suggested that the signal peptide had been processed. We also constructed a series of plasmids in which the phaseolin signal-peptide-coding region was either removed or replaced with increasing amounts of the amino-terminal coding region for acid phosphatase. Phaseolin polypeptides with no signal peptide were not posttranslationally modified in S. cerevisiae. Partial or complete substitution of the phaseolin signal peptide with that from acid phosphatase dramatically inhibited both signal peptide processing and glycosylation, suggesting that some specific feature of the phaseolin signal amino acid sequence was required for these modifications to occur. Larger hybrid proteins that included approximately one-half of the acid phosphatase sequence linked to the amino terminus of the mature phaseolin polypeptide did undergo proteolytic processing and glycosylation. However, these polypeptides were cleaved at several sites that are not normally used in the unaltered acid phosphatase protein.

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Dimerization of the RamC Morphogenetic Protein of Streptomyces coelicolor

Journal of Bacteriology ◽

10.1128/jb.186.5.1330-1336.2004 ◽

2004 ◽

Vol 186 (5) ◽

pp. 1330-1336 ◽

Cited By ~ 5

Author(s):

Michael E. Hudson ◽

Justin R. Nodwell

Keyword(s):

Protein Kinase ◽

Streptomyces Coelicolor ◽

Kinase Domain ◽

Terminal Protein ◽

Amino Terminal ◽

Morphogenetic Protein ◽

Aerial Hyphae ◽

Multiple Copies ◽

Defective Allele

ABSTRACT RamC is required for the formation of spore-forming cells called aerial hyphae by the bacterium Streptomyces coelicolor. This protein is membrane associated and has an amino-terminal protein kinase-like domain, but little is known about its mechanism of action. In this study we found that the presence of multiple copies of a defective allele of ramC inhibits morphogenesis in S. coelicolor, consistent with either titration of a target or formation of inactive RamC multimers. We identified a domain in RamC that is C terminal to the putative kinase domain and forms a dimer with a Kd of ∼0.1 μM. These data suggest that RamC acts as a dimer in vivo.

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Isolation and DNA sequence of ADH3, a nuclear gene encoding the mitochondrial isozyme of alcohol dehydrogenase in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.5.11.3024-3034.1985 ◽

1985 ◽

Vol 5 (11) ◽

pp. 3024-3034

Author(s):

E T Young ◽

D Pilgrim

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acid ◽

Nucleotide Sequence ◽

Alcohol Dehydrogenase ◽

Nuclear Gene ◽

Amino Acid Identity ◽

Leader Peptide ◽

Nucleotide Sequence Analysis ◽

Amino Terminal ◽

Cloned Gene

The Saccharomyces cerevisiae nuclear gene, ADH3, that encodes the mitochondrial alcohol dehydrogenase isozyme ADH III was cloned by virtue of its nucleotide homology to ADH1 and ADH2. Both chromosomal and plasmid-encoded ADH III isozymes were repressed by glucose and migrated heterogeneously on nondenaturing gels. Nucleotide sequence analysis indicated 73 and 74% identity for ADH3 with ADH1 and ADH2, respectively. The amino acid identity between the predicted ADH III polypeptide and ADH I and ADH II was 79 and 80%, respectively. The open reading frame encoding ADH III has a highly basic 27-amino-acid amino-terminal extension relative to ADH I and ADH II. The nucleotide sequence of the presumed leader peptide has a high degree of identity with the untranslated leader regions of ADH1 and ADH2 mRNAs. A strain containing a null allele of ADH3 did not have a detectably altered phenotype. The cloned gene integrated at the ADH3 locus, indicating that this is the structural gene for ADH III.

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Analysis of the essential and excision repair functions of the RAD3 gene of Saccharomyces cerevisiae by mutagenesis

Molecular and Cellular Biology ◽

10.1128/mcb.6.4.1218-1227.1986 ◽

1986 ◽

Vol 6 (4) ◽

pp. 1218-1227

Author(s):

L Naumovski ◽

E C Friedberg

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Binding ◽

Excision Repair ◽

Random Mutagenesis ◽

Nucleotide Binding ◽

Yeast Cells ◽

Wild Type ◽

Binding Region ◽

Site Specific ◽

Essential Function

The RAD3 gene of Saccharomyces cerevisiae, which is involved in excision repair of DNA and is essential for cell viability, was mutagenized by site-specific and random mutagenesis. Site-specific mutagenesis was targeted to two regions near the 5' and 3' ends of the coding region, selected on the basis of amino acid sequence homology with known nucleotide binding and with known specific DNA-binding proteins, respectively. Two mutations in the putative nucleotide-binding region and one in the putative DNA-binding region inactivate the excision repair function of the gene, but not the essential function. A gene encoding two tandem mutations in the putative DNA-binding region is defective in both excision repair and essential functions of RAD3. Seven plasmids were isolated following random mutagenesis with hydroxylamine. Mutations in six of these plasmids were identified by gap repair of mutant plasmids from the chromosome of strains with previously mapped rad3 mutations, followed by DNA sequencing. Three of these contain missense mutations which inactivate only the excision repair function. The other three carry nonsense mutations which inactivate both the excision repair and essential functions. Collectively our results indicate that the RAD3 excision repair function is more sensitive to inactivation than is the essential function. Overexpression of wild-type Rad3 protein and a number of rad3 mutant proteins did not affect the UV resistance of wild-type yeast cells. However, overexpression of Rad3-2 protein rendered wild-type cells partially UV sensitive, indicating that excess Rad3-2 protein is dominant to the wild-type form. These and other results suggest that Rad3-2 protein retains its affinity for damaged DNA or other substrates, but is not catalytically active in excision repair.

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