Expression of the 180-kD Ribosome Receptor Induces Membrane Proliferation and Increased Secretory Activity in Yeast

Expression of the canine 180-kD ribosome receptor (p180) in yeast cells resulted in a marked proliferation of intracellular membranes. The type of membranes observed varied with the expression of specific portions of p180. Rough membranes predominated when the ribosome binding domain of p180 was present, whereas expression constructs lacking this region resulted in smooth membranes. Northern analysis indicated that expression of the NH2-terminal 767 amino acids (ΔCT), which include the ribosome binding domain, upregulated the transcription and translation of genes involved in exocytosis. The membranes that were proliferated were functional as these cells overcame a temperature-sensitive translocation defect. Most significantly, cells that overexpressed ΔCT and proliferated rough endoplasmic reticulum exhibited severalfold higher levels of secretion of an ectopically expressed secretory protein. We conclude that p180 expression triggers a cascade of events leading to an increase in secretory potential akin to the terminal differentiation of mammalian secretory cells and tissues.

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Important Role for Phylogenetically Invariant PP2Acα Active Site and C-Terminal Residues Revealed by Mutational Analysis in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/156.1.21 ◽

2000 ◽

Vol 156 (1) ◽

pp. 21-29 ◽

Cited By ~ 1

Author(s):

David R H Evans ◽

Brian A Hemmings

Keyword(s):

Protein Interactions ◽

Active Site ◽

Protein Function ◽

Mutational Analysis ◽

Yeast Cells ◽

Temperature Sensitive ◽

Active Site Residues ◽

Catalytic Function

Abstract PP2A is a central regulator of eukaryotic signal transduction. The human catalytic subunit PP2Acα functionally replaces the endogenous yeast enzyme, Pph22p, indicating a conservation of function in vivo. Therefore, yeast cells were employed to explore the role of invariant PP2Ac residues. The PP2Acα Y127N substitution abolished essential PP2Ac function in vivo and impaired catalysis severely in vitro, consistent with the prediction from structural studies that Tyr-127 mediates substrate binding and its side chain interacts with the key active site residues His-118 and Asp-88. The V159E substitution similarly impaired PP2Acα catalysis profoundly and may cause global disruption of the active site. Two conditional mutations in the yeast Pph22p protein, F232S and P240H, were found to cause temperature-sensitive impairment of PP2Ac catalytic function in vitro. Thus, the mitotic and cell lysis defects conferred by these mutations result from a loss of PP2Ac enzyme activity. Substitution of the PP2Acα C-terminal Tyr-307 residue by phenylalanine impaired protein function, whereas the Y307D and T304D substitutions abolished essential function in vivo. Nevertheless, Y307D did not reduce PP2Acα catalytic activity significantly in vitro, consistent with an important role for the C terminus in mediating essential protein-protein interactions. Our results identify key residues important for PP2Ac function and characterize new reagents for the study of PP2A in vivo.

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180-kD ribosome receptor is essential for both ribosome binding and protein translocation.

The Journal of Cell Biology ◽

10.1083/jcb.120.4.853 ◽

1993 ◽

Vol 120 (4) ◽

pp. 853-863 ◽

Cited By ~ 38

Author(s):

A J Savitz ◽

D I Meyer

Keyword(s):

Monoclonal Antibodies ◽

Direct Evidence ◽

Protein Translocation ◽

Secretory Protein ◽

Ribosome Binding ◽

Fab Fragments ◽

Rough Er

We have previously isolated a 180-kD ribosome receptor (p180) from mammalian rough ER that, when incorporated into liposomes, bound ribosomes with an affinity similar to intact membranes. To directly assess the contribution of p180 to ribosome binding as well as protein translocation, monoclonal antibodies were used to selectively deplete p180 from the detergent extracts of rough ER membranes used in the preparation of translocation-competent proteoliposomes. Proteoliposomes prepared from p180-depleted extracts showed a reduction in ribosome binding to the level of trypsin-inactivated controls as well as a loss in their ability to cotranslationally translocate two different secretory protein precursors. When purified p180 was added back to depleted extracts before proteoliposome formation, both ribosome binding and translocation activity were restored. In addition, the monoclonal antibodies, as well as their Fab' fragments, were able to inhibit ribosome binding and protein translocation when bound to intact rough microsomes. These data provide direct evidence that the 180-kD ribosome receptor is essential for ribosome binding and for the translocation of nascent proteins across the membrane of the rough ER.

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Schizosaccharomyces pombe ras1 and byr1 are functionally related genes of the ste family that affect starvation-induced transcription of mating-type genes.

Molecular and Cellular Biology ◽

10.1128/mcb.10.2.549 ◽

1990 ◽

Vol 10 (2) ◽

pp. 549-560 ◽

Cited By ~ 43

Author(s):

S A Nadin-Davis ◽

A Nasim

Keyword(s):

Gene Family ◽

Fission Yeast ◽

Mating Type ◽

Growth Cycle ◽

Yeast Cells ◽

Null Alleles ◽

Northern Analysis ◽

Gene Transcript ◽

Type Gene

We have further investigated the function of the ras1 and byr1 genes, which were previously shown to be critical for sexual differentiation in fission yeast cells. Several physiological similarities between strains containing null alleles of these genes supports the idea that ras1 and byr1 are functionally closely related. Furthermore, we have found that byr1 is allelic to ste1, one of at least 10 genes which when mutated can cause sterility. Since ras1 had previously been found to be allelic to ste5, both ras and byr genes are now clearly shown to be a part of the ste gene family, thus confirming their close functional relationship. The observation that the mating-type loci could overcome the sporulation block of ras1 and byr1 mutant strains prompted investigation of the role of the ras-byr pathway in the induction of the mating-type gene transcripts upon nitrogen starvation. By Northern analysis of RNA preparations from strains carrying wild-type or mutant ras1 alleles and grown to different stages of the growth cycle, we have shown that ras1 plays an important role in inducing the Pi transcript of the mating-type loci and the mei3 gene transcript. These observations provide a molecular basis for the role of the ste gene family, including ras1 and byr1, in meiosis and indicate that further characterization of other ste genes would be very useful for elucidating the mechanism of ras1 function in fission yeast cells.

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The role of RAP1 in the regulation of the MAT alpha locus

Molecular and Cellular Biology ◽

10.1128/mcb.11.2.1069-1079.1991 ◽

1991 ◽

Vol 11 (2) ◽

pp. 1069-1079

Author(s):

D Giesman ◽

L Best ◽

K Tatchell

Keyword(s):

Transcriptional Activation ◽

Consensus Sequence ◽

Point Mutations ◽

Yeast Cells ◽

Effective Dosage ◽

Upstream Region ◽

Yeast Genome ◽

Temperature Sensitive ◽

Lacz Gene

The RAP1 gene of Saccharomyces cerevisiae encodes an abundant DNA-binding protein, also known as GRF1, TBA, or TUF, that binds to many sites in the yeast genome in vitro. These sites define a consensus sequence, [sequence: see text], and deletion analyses of genes that contain this sequence have implicated the involvement of RAP1 in numerous cellular processes, including gene activation and repression. The MAT alpha locus, required for determination of the alpha cell type in yeast cells, contains a RAP1 binding site; this site coincides with the MAT alpha upstream activating sequence (UAS) and is necessary for expression of the two genes encoded by the MAT alpha locus, MAT alpha 1 and MAT alpha 2. We show that the MAT alpha UAS is sufficient to activate transcription from a promoterless gene fusion of the yeast CYC1 upstream region and the lacZ gene. Constructs containing only the MAT alpha UAS generated elevated levels of beta-galactosidase activity which were indistinguishable from those of constructs containing the entire MAT alpha intergenic region. Further, the MAT alpha UAS has an intrinsic polarity of transcriptional activation; transcription of CYC1-lacZ was six- to sevenfold higher when the UAS was oriented in the direction normally associated with MAT alpha 2 transcription. Point mutations in the MAT alpha UAS that reduce MAT alpha expression three- to fivefold resulted in a bi-mating phenotype, while a mutation that reduced MAT alpha expression still further resulted in an a-mating phenotype. We isolated plasmids from a high-copy-number yeast library that suppressed the bi-mating defect of point mutations in the MAT alpha UAS, and the most effective dosage suppressor contained the gene encoding RAP1. A temperature-sensitive rap1 mutant bi-mates at the semipermissive temperature. Double mutants at rap1 and mat alpha mate exclusively as a cells, at all temperatures, and do not express detectable levels of MAT alpha RNA. These data provide evidence that the RAP1 gene product functions at the MAT alpha UAS in vivo.

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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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MKK1 and MKK2, which encode Saccharomyces cerevisiae mitogen-activated protein kinase-kinase homologs, function in the pathway mediated by protein kinase C

Molecular and Cellular Biology ◽

10.1128/mcb.13.5.3076-3083.1993 ◽

1993 ◽

Vol 13 (5) ◽

pp. 3076-3083

Author(s):

K Irie ◽

M Takase ◽

K S Lee ◽

D E Levin ◽

H Araki ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Kinase C ◽

Protein Kinase ◽

Map Kinase ◽

Protein Kinases ◽

Cell Lysis ◽

Yeast Cells ◽

Temperature Sensitive ◽

Kinase C ◽

Mitogen Activated Protein

The PKC1 gene of Saccharomyces cerevisiae encodes a homolog of mammalian protein kinase C that is required for normal growth and division of yeast cells. We report here the isolation of the yeast MKK1 and MKK2 (for mitogen-activated protein [MAP] kinase-kinase) genes which, when overexpressed, suppress the cell lysis defect of a temperature-sensitive pkc1 mutant. The MKK genes encode protein kinases most similar to the STE7 product of S. cerevisiae, the byr1 product of Schizosaccharomyces pombe, and vertebrate MAP kinase-kinases. Deletion of either MKK gene alone did not cause any apparent phenotypic defects, but deletion of both MKK1 and MKK2 resulted in a temperature-sensitive cell lysis defect that was suppressed by osmotic stabilizers. This phenotypic defect is similar to that associated with deletion of the BCK1 gene, which is thought to function in the pathway mediated by PCK1. The BCK1 gene also encodes a predicted protein kinase. Overexpression of MKK1 suppressed the growth defect caused by deletion of BCK1, whereas an activated allele of BCK1 (BCK1-20) did not suppress the defect of the mkk1 mkk2 double disruption. Furthermore, overexpression of MPK1, which encodes a protein kinase closely related to vertebrate MAP kinases, suppressed the defect of the mkk1 mkk2 double mutant. These results suggest that MKK1 and MKK2 function in a signal transduction pathway involving the protein kinases encoded by PKC1, BCK1, and MPK1. Genetic epistasis experiments indicated that the site of action for MKK1 and MKK2 is between BCK1 and MPK1.

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U4 small nuclear RNA dissociates from a yeast spliceosome and does not participate in the subsequent splicing reaction.

Molecular and Cellular Biology ◽

10.1128/mcb.11.11.5571 ◽

1991 ◽

Vol 11 (11) ◽

pp. 5571-5577 ◽

Cited By ~ 45

Author(s):

S L Yean ◽

R J Lin

Keyword(s):

Mrna Splicing ◽

Yeast Cells ◽

Temperature Sensitive ◽

Small Nuclear Rna ◽

Small Nuclear Rnas ◽

Nuclear Rna ◽

Sensitive Allele ◽

U4 Rna ◽

Small Nuclear Ribonucleoproteins

U4 and U6 small nuclear RNAs reside in a single ribonucleoprotein particle, and both are required for pre-mRNA splicing. The U4/U6 and U5 small nuclear ribonucleoproteins join U1 and U2 on the pre-mRNA during spliceosome assembly. Binding of U4 is then destabilized prior to or concomitant with the 5' cleavage-ligation. In order to test the role of U4 RNA, we isolated a functional spliceosome by using extracts prepared from yeast cells carrying a temperature-sensitive allele of prp2 (rna2). The isolated prp2 delta spliceosome contains U2, U5, U6, and possibly also U1 and can be activated to splice the bound pre-mRNA. U4 RNA does not associate with the isolated spliceosomes and is shown not to be involved in the subsequent cleavage-ligation reactions. These results are consistent with the hypothesis that the role of U4 in pre-mRNA splicing is to deliver U6 to the spliceosome.

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U4 small nuclear RNA dissociates from a yeast spliceosome and does not participate in the subsequent splicing reaction

Molecular and Cellular Biology ◽

10.1128/mcb.11.11.5571-5577.1991 ◽

1991 ◽

Vol 11 (11) ◽

pp. 5571-5577

Author(s):

S L Yean ◽

R J Lin

Keyword(s):

Mrna Splicing ◽

Yeast Cells ◽

Temperature Sensitive ◽

Small Nuclear Rna ◽

Small Nuclear Rnas ◽

Nuclear Rna ◽

Sensitive Allele ◽

U4 Rna ◽

Small Nuclear Ribonucleoproteins

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New Mutants of Saccharomyces cerevisiae Affected in the Transport of Proteins From the Endoplasmic Reticulum to the Golgi Complex

Genetics ◽

10.1093/genetics/142.2.393 ◽

1996 ◽

Vol 142 (2) ◽

pp. 393-406 ◽

Cited By ~ 11

Author(s):

Linda J Wuestehube ◽

Rainer Duden ◽

Arlene Eun ◽

Susan Hamamoto ◽

Paul Korn ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Golgi Complex ◽

Secretory Pathway ◽

Secretory Protein ◽

Temperature Sensitive ◽

Nonpermissive Temperature ◽

Membrane Structures ◽

Α Subunit ◽

Complementation Groups ◽

Vacuolar Protein

Abstract We have isolated new temperature-sensitive mutations in five complementation groups, sec31-sec35, that are defective in the transport of proteins from the endoplasmic reticulum (ER) to the Golgi complex. The sec31-sec35 mutants and additional alleles of previously identified sec and vacuolar protein sorting (vps) genes were isolated in a screen based on the detection of α-factor precursor in yeast colonies replicated to and lysed on nitrocellulose filters. Secretory protein precursors accumulated in sec31-sec35 mutants at the nonpermissive temperature were core-glycosylated but lacked outer chain carbohydrate, indicating that transport was blocked after translocation into the ER but before arrival in the Golgi complex. Electron microscopy revealed that the newly identified sec mutants accumulated vesicles and membrane structures reminiscent of secretory pathway organelles. Complementation analysis revealed that sec32-1 is an allele of BOS1, a gene implicated in vesicle targeting to the Golgi complex, and sec33-1 is an allele of RET1, a gene that encodes the α subunit of coatomer.

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ΔLys120, a mutation which destabilizes the ribosome-binding domain of ribosomal protein L7/L12

Biochemistry and Cell Biology ◽

10.1139/o90-123 ◽

1990 ◽

Vol 68 (5) ◽

pp. 832-838 ◽

Cited By ~ 1

Author(s):

Michael Laughrea ◽

Eric Higgins

Keyword(s):

Protein Synthesis ◽

Ribosomal Protein ◽

Binding Domain ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Ribosome Binding ◽

Mutant Genes

Five-residue-long deletions centered on A1a63, A1a75, and Glu118 of ribosomal protein L7/L12 gave low mutant yields (5% or less) when the mutant genes were cloned in phage M13mp18 and controlled by the L10 promotor. Deletions of Glu118–Lys120 or Lys120 (the COOH-terminus of L7/L12) gave higher mutant yields, up to 50% with L7/L12ΔLys120. L7/L12ΔLys120 was not preferentially found in the S100 and not preferentially removed by LiCl washing, but was preferentially extracted from 70S ribosomes in the presence of 28–35% ethanol in 0.25–0.5 M NH4Cl. It follows that ΔLys120 destabilizes the ribosome-binding domain of ribosomal protein L7/L12 in an ethanol-containing solvent, which raises the question whether Lys120 is part of the ribosome-binding domain of L7/L12 during some step of protein synthesis or whether it is essential to preserve the conformation of the physiological ribosome-binding domain under structurally stressful conditions.Key words: ribosome, ribosomal protein L7/L12, site-directed mutagenesis.

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