scholarly journals The yeast SEN3 gene encodes a regulatory subunit of the 26S proteasome complex required for ubiquitin-dependent protein degradation in vivo.

1995 ◽  
Vol 15 (11) ◽  
pp. 6311-6321 ◽  
Author(s):  
D J DeMarini ◽  
F R Papa ◽  
S Swaminathan ◽  
D Ursic ◽  
T P Rasmussen ◽  
...  
Keyword(s):  
Sequence Similarity ◽  
Genetic Selection ◽  
26S Proteasome ◽  
Yeast Cells ◽  
Regulatory Subunit ◽  
20S Proteasome ◽  
Amino Terminal ◽  
A Subunit

The yeast Sen1 protein was discovered by virtue of its role in tRNA splicing in vitro. To help determine the role of Sen1 in vivo, we attempted to overexpress the protein in yeast cells. However, cells with a high-copy SEN1-bearing plasmid, although expressing elevated amounts of SEN1 mRNA, show little increase in the level of the encoded protein, indicating that a posttranscriptional mechanism limits SEN1 expression. This control depends on an amino-terminal element of Sen1. Using a genetic selection for mutants with increased expression of Sen1-derived fusion proteins, we identified mutations in a novel gene, designated SEN3. SEN3 is essential and encodes a 945-residue protein with sequence similarity to a subunit of an activator of the 20S proteasome from bovine erythrocytes, called PA700. Earlier work indicated that the 20S proteasome associates with a multisubunit regulatory factor, resulting in a 26S proteasome complex that degrades substrates of the ubiquitin system. Mutant sen3-1 cells have severe defects in the degradation of such substrates and accumulate ubiquitin-protein conjugates. Most importantly, we show biochemically that Sen3 is a subunit of the 26S proteasome. These data provide evidence for the involvement of the 26S proteasome in the degradation of ubiquitinated proteins in vivo and for a close relationship between PA700 and the regulatory complexes within the 26S proteasome, and they directly demonstrate that Sen3 is a component of the yeast 26S proteasome.

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

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.

Control of AMP deaminase 1 binding to myosin heavy chain

1998 ◽  
Vol 275 (3) ◽  
pp. C870-C881 ◽  
Author(s):  
Ichiro Hisatome ◽  
Takayuki Morisaki ◽  
Hiroshi Kamma ◽  
Takako Sugama ◽  
Hiroko Morisaki ◽  
...  
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Sequence Similarity ◽  
Critical Role ◽  
Energy Charge ◽  
Adenylate Energy Charge ◽  
Amp Deaminase ◽  
Amino Terminal

AMP deaminase (AMPD) plays a central role in preserving the adenylate energy charge in myocytes following exercise and in producing intermediates for the citric acid cycle in muscle. Prior studies have demonstrated that AMPD1 binds to myosin heavy chain (MHC) in vitro; binding to the myofibril varies with the state of muscle contraction in vivo, and binding of AMPD1 to MHC is required for activation of this enzyme in myocytes. The present study has identified three domains in AMPD1 that influence binding of this enzyme to MHC using a cotransfection model that permits assessment of mutations introduced into the AMPD1 peptide. One domain that encompasses residues 178–333 of this 727-amino acid peptide is essential for binding of AMPD1 to MHC. This region of AMPD1 shares sequence similarity with several regions of titin, another MHC binding protein. Two additional domains regulate binding of this peptide to MHC in response to intracellular and extracellular signals. A nucleotide binding site, which is located at residues 660–674, controls binding of AMPD1 to MHC in response to changes in intracellular ATP concentration. Deletion analyses demonstrate that the amino-terminal 65 residues of AMPD1 play a critical role in modulating the sensitivity to ATP-induced inhibition of MHC binding. Alternative splicing of the AMPD1 gene product, which alters the sequence of residues 8–12, produces two AMPD1 isoforms that exhibit different MHC binding properties in the presence of ATP. These findings are discussed in the context of the various roles proposed for AMPD in energy production in the myocyte.

scholarly journals Cloning and Characterization of TaSAP7-A, a Member of the Stress-Associated Protein Family in Common Wheat

2021 ◽  
Vol 12 ◽  
Author(s):  
Wenlu Li ◽  
Yixue Wang ◽  
Runzhi Li ◽  
Xiaoping Chang ◽  
Xiangyang Yuan ◽  
...  
Keyword(s):  
Abiotic Stress ◽  
Chlorophyll Content ◽  
Agronomic Traits ◽  
Abiotic Stress Tolerance ◽  
Grain Filling ◽  
26S Proteasome ◽  
Regulatory Subunit ◽  
Germination Stage

Stress association proteins (SAPs) are A20/AN1 zinc-finger domain proteins, which play important roles in plant adaptation to abiotic stress and plant development. The functions of SAPs in some plants were reported, but little is known about it in wheat (Triticum aestivum L.). In this study, we characterized a novel 2AN1-type stress association protein gene TaSAP7-A, which was mapped to chromosome 5A in wheat. Subcellular localization indicated that TaSAP7-A was distributed in the nucleus and cytoplasm. Unlike previously known A20/AN1-type SAP genes, TaSAP7-A was negatively regulated to abiotic stress tolerance. Overexpressing TaSAP7-A Arabidopsis lines were hypersensitive to ABA, osmotic and salt stress at germination stage and post-germination stage. Overexpression of TaSAP7-A Arabidopsis plants accelerated the detached leaves’ chlorophyll degradation. Association analysis of TaSAP7-A haplotypes and agronomic traits showed that Hap-5A-2 was significantly associated with higher chlorophyll content at jointing stage and grain-filling stage. These results jointly revealed that TaSAP7-A is related to the chlorophyll content in the leaves of Arabidopsis and wheat. Both in vivo and in vitro experiments demonstrated that TaSAP7-A interacted with TaS10B, which was the component of regulatory subunit in 26S proteasome. In general, TaSAP7-A was a regulator of chlorophyll content, and favorable haplotypes should be helpful for improving plant chlorophyll content and grain yield of wheat.

scholarly journals Regulatory subunit (CNB1 gene product) of yeast Ca2+/calmodulin-dependent phosphoprotein phosphatases is required for adaptation to pheromone.

1992 ◽  
Vol 12 (8) ◽  
pp. 3460-3469 ◽  
Author(s):  
M S Cyert ◽  
J Thorner
Keyword(s):  
Yeast Cells ◽  
Regulatory Subunit ◽  
Expression Library ◽  
Yeast Saccharomyces Cerevisiae ◽  
B Subunit ◽  
Prominent Component ◽  
Genes Encoding ◽  
Yeast Genes

By using an assay specific for detection of calcineurin, a Ca2+/calmodulin-dependent phosphoprotein phosphatase, this enzyme was purified approximately 5,000-fold from extracts of the yeast Saccharomyces cerevisiae. Cna1p and Cna2p, the products of two yeast genes encoding the catalytic (A) subunits of calcineurin, were major constituents of the purified fraction. A third prominent component of apparent molecular mass 16 kDa displayed several properties, including ability to bind 45Ca2+, that are characteristic of the regulatory (B) subunit of mammalian calcineurin and was recognized by an antiserum raised against bovine calcineurin. These antibodies were used to isolate the structural gene (CNB1) encoding this protein from a yeast expression library in the vector lambda gt11. The nucleotide sequence of CNB1 predicted a polypeptide similar in length and highly related in amino acid sequence (56% identity) to the mammalian calcineurin B subunit. Like its counterpart in higher cells, yeast Cnb1p was myristoylated at its N terminus. Mutants lacking Cnb1p, or all three calcineurin subunits (Cna1p, Cna2p, and Cnb1p), were viable. Extracts of cnb1 delta mutants contained no detectable calcineurin activity, even though Cna1p and Cna2p were present at normal levels, suggesting that the B subunit is required for full enzymatic activity in vitro. As was observed previously for MATa cna1 cna2 double mutants, MATa cnb1 mutants were defective in their ability to recover from alpha-factor-induced growth arrest. Thus, the B subunit also is required for the function of calcineurin in promoting adaptation of haploid yeast cells to pheromone in vivo.

scholarly journals Identification and Characterization of a Novel Evolutionarily Conserved Lysine-specific Methyltransferase Targeting Eukaryotic Translation Elongation Factor 2 (eEF2)

2014 ◽  
Vol 289 (44) ◽  
pp. 30499-30510 ◽  
Author(s):  
Erna Davydova ◽  
Angela Y. Y. Ho ◽  
Jedrzej Malecki ◽  
Anders Moen ◽  
Jorrit M. Enserink ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Sequence Similarity ◽  
Elongation Factor ◽  
Protein Translation ◽  
Cellular Protein ◽  
Yeast Cells ◽  
Lysine Methylation ◽  
Elongation Factor 2

The components of the cellular protein translation machinery, such as ribosomal proteins and translation factors, are subject to numerous post-translational modifications. In particular, this group of proteins is frequently methylated. However, for the majority of these methylations, the responsible methyltransferases (MTases) remain unknown. The human FAM86A (family with sequence similarity 86) protein belongs to a recently identified family of protein MTases, and we here show that FAM86A catalyzes the trimethylation of eukaryotic elongation factor 2 (eEF2) on Lys-525. Moreover, we demonstrate that the Saccharomyces cerevisiae MTase Yjr129c, which displays sequence homology to FAM86A, is a functional FAM86A orthologue, modifying the corresponding residue (Lys-509) in yeast eEF2, both in vitro and in vivo. Finally, Yjr129c-deficient yeast cells displayed phenotypes related to eEF2 function (i.e. increased frameshifting during protein translation and hypersensitivity toward the eEF2-specific drug sordarin). In summary, the present study establishes the function of the previously uncharacterized MTases FAM86A and Yjr129c, demonstrating that these enzymes introduce a functionally important lysine methylation in eEF2. Based on the previous naming of similar enzymes, we have redubbed FAM86A and Yjr129c as eEF2-KMT and Efm3, respectively.

Regulatory subunit (CNB1 gene product) of yeast Ca2+/calmodulin-dependent phosphoprotein phosphatases is required for adaptation to pheromone

1992 ◽  
Vol 12 (8) ◽  
pp. 3460-3469
Author(s):  
M S Cyert ◽  
J Thorner
Keyword(s):  
Yeast Cells ◽  
Regulatory Subunit ◽  
Expression Library ◽  
Yeast Saccharomyces Cerevisiae ◽  
B Subunit ◽  
Prominent Component ◽  
Genes Encoding ◽  
Yeast Genes

By using an assay specific for detection of calcineurin, a Ca2+/calmodulin-dependent phosphoprotein phosphatase, this enzyme was purified approximately 5,000-fold from extracts of the yeast Saccharomyces cerevisiae. Cna1p and Cna2p, the products of two yeast genes encoding the catalytic (A) subunits of calcineurin, were major constituents of the purified fraction. A third prominent component of apparent molecular mass 16 kDa displayed several properties, including ability to bind 45Ca2+, that are characteristic of the regulatory (B) subunit of mammalian calcineurin and was recognized by an antiserum raised against bovine calcineurin. These antibodies were used to isolate the structural gene (CNB1) encoding this protein from a yeast expression library in the vector lambda gt11. The nucleotide sequence of CNB1 predicted a polypeptide similar in length and highly related in amino acid sequence (56% identity) to the mammalian calcineurin B subunit. Like its counterpart in higher cells, yeast Cnb1p was myristoylated at its N terminus. Mutants lacking Cnb1p, or all three calcineurin subunits (Cna1p, Cna2p, and Cnb1p), were viable. Extracts of cnb1 delta mutants contained no detectable calcineurin activity, even though Cna1p and Cna2p were present at normal levels, suggesting that the B subunit is required for full enzymatic activity in vitro. As was observed previously for MATa cna1 cna2 double mutants, MATa cnb1 mutants were defective in their ability to recover from alpha-factor-induced growth arrest. Thus, the B subunit also is required for the function of calcineurin in promoting adaptation of haploid yeast cells to pheromone in vivo.

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

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.

scholarly journals Rsp5 Ubiquitin-Protein Ligase Mediates DNA Damage-Induced Degradation of the Large Subunit of RNA Polymerase II in Saccharomyces cerevisiae

1999 ◽  
Vol 19 (10) ◽  
pp. 6972-6979 ◽  
Author(s):  
Sylvie L. Beaudenon ◽  
Maria R. Huacani ◽  
Guangli Wang ◽  
Donald P. McDonnell ◽  
Jon M. Huibregtse
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Large Subunit ◽  
26S Proteasome ◽  
Regulatory Subunit ◽  
Ubiquitin Protein Ligase

ABSTRACT Rsp5 is an E3 ubiquitin-protein ligase of Saccharomyces cerevisiae that belongs to the hect domain family of E3 proteins. We have previously shown that Rsp5 binds and ubiquitinates the largest subunit of RNA polymerase II, Rpb1, in vitro. We show here that Rpb1 ubiquitination and degradation are induced in vivo by UV irradiation and by the UV-mimetic compound 4-nitroquinoline-1-oxide (4-NQO) and that a functional RSP5 gene product is required for this effect. The 26S proteasome is also required; a mutation ofSEN3/RPN2 (sen3-1), which encodes an essential regulatory subunit of the 26S proteasome, partially blocks 4-NQO-induced degradation of Rpb1. These results suggest that Rsp5-mediated ubiquitination and degradation of Rpb1 are components of the response to DNA damage. A human WW domain-containing hect (WW-hect) E3 protein closely related to Rsp5, Rpf1/hNedd4, also binds and ubiquitinates both yeast and human Rpb1 in vitro, suggesting that Rpf1 and/or another WW-hect E3 protein mediates UV-induced degradation of the large subunit of polymerase II in human cells.

scholarly journals Gpi19, the Saccharomyces cerevisiae Homologue of Mammalian PIG-P, Is a Subunit of the Initial Enzyme for Glycosylphosphatidylinositol Anchor Biosynthesis

2005 ◽  
Vol 4 (11) ◽  
pp. 1801-1807 ◽  
Author(s):  
Heather A. Newman ◽  
Martin J. Romeo ◽  
Sarah E. Lewis ◽  
Benjamin C. Yan ◽  
Peter Orlean ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Sequence Similarity ◽  
Secretory Proteins ◽  
Temperature Sensitive ◽  
Transferase Activity ◽  
Its Gene ◽  
A Subunit ◽  
Glcnac Transferase

ABSTRACT Glycosylphosphatidylinositols (GPIs) are attached to the C termini of some glycosylated secretory proteins, serving as membrane anchors for many of those on the cell surface. Biosynthesis of GPIs is initiated by the transfer of N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to phosphatidylinositol. This reaction is carried out at the endoplasmic reticulum (ER) by an enzyme complex called GPI-N-acetylglucosaminyltransferase (GPI-GlcNAc transferase). The human enzyme has six known subunits, at least four of which, GPI1, PIG-A, PIG-C, and PIG-H, have functional homologs in the budding yeast Saccharomyces cerevisiae. The uncharacterized yeast gene YDR437w encodes a protein with some sequence similarity to human PIG-P, a fifth subunit of the GPI-GlcNAc transferase. Here we show that Ydr437w is a small but essential subunit of the yeast GPI-GlcNAc transferase, and we designate its gene GPI19. Similar to other mutants in the yeast enzyme, temperature-sensitive gpi19 mutants display cell wall defects and hyperactive Ras phenotypes. The Gpi19 protein associates with the yeast GPI-GlcNAc transferase in vivo, as judged by coimmuneprecipitation with the Gpi2 subunit. Moreover, conditional gpi19 mutants are defective for GPI-GlcNAc transferase activity in vitro. Finally, we present evidence for the topology of Gpi19 within the ER membrane.

scholarly journals PSMC2/E2F1 Axis Promotes the Development and Progression of Glioma

2021 ◽  
Author(s):  
Xuchen Qi ◽  
Qin Lu ◽  
Dajiang Xie ◽  
Junhui Lv ◽  
Yiwei Liu ◽  
...  
Keyword(s):  
Glioma Cells ◽  
26S Proteasome ◽  
Cell Counting ◽  
Regulatory Subunit ◽  
Downstream Target ◽  
Normal Tissues ◽  
E2f Transcription Factor ◽  
And Migration

Abstract Background Glioma is the most common type of malignant brain tumor with limited treatment strategy and poor prognosis. Proteasome 26S subunit ATPase 2 (PSMC2) is a key member of the 26S proteasome 19S regulatory subunit, whose role in glioma is still not clear. In this context, we aim to explore the role of PSMC2 in glioma in vitro and in vivo. Methods Expression of PSMC2 in in glioma tissues and normal tissues are detected by immunohistochemical (IHC) analysis. The proliferation assays (MTT assay and Celigo cell counting assay), flow cytometry and migration assays (wound-healing assay and Transwell) are used to detect the effects of PSMC2 knockdown on glioma cells. The influence of PSMC2 knockdown on tumor growth in vivo was evaluated by mice xenograft models. In addition, the downstream target of PSMC2 is determined by human GeneChip and Ingenuity Pathway Analysis (IPA). Results PSMC2 is overexpressed in glioma tissues than normal tissues. Moreover, knockdown of PSMC2 can inhibit the proliferation, migration and arrest cell cycle in G2 phase of glioma cells. Additionally, PSMC2 knockdown promotes glioma cell apoptosis by increasing expression of caspase3, caspase8, IGFBP-1, while reducing expression of IGF-I, Survivin, TRAILR-4. In vivo findings reveal that PSMC2 knockdown inhibit the tumorigenicity of glioma cells. Furthermore, downstream of PSMC2 is explored, identifying E2F transcription factor 1 (E2F1) as a potential target. Notably, E2F1 knockdown exhibits similar effects on the development of glioma with PSMC2, which could strengthen the inhibition effects of PSMC2 knockdown on glioma synergistically. Conclusions PSMC2 is closely associated with glioma development by targeting E2F1, and might be considered as a novel therapeutic target in patients with glioma.

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