scholarly journals Recognition of nonproline N-terminal residues by the Pro/N-degron pathway

2020 ◽  
Vol 117 (25) ◽  
pp. 14158-14167 ◽  
Author(s):  
Cheng Dong ◽  
Shun-Jia Chen ◽  
Artem Melnykov ◽  
Sara Weirich ◽  
Kelly Sun ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Crystal Structures ◽  
Ubiquitin Ligase ◽  
Substrate Recognition ◽  
26S Proteasome ◽  
Yeast Cells ◽  
Sequence Motifs ◽  
Wild Type ◽  
Rapid Degradation ◽  
A Subunit

Eukaryotic N-degron pathways are proteolytic systems whose unifying feature is their ability to recognize proteins containing N-terminal (Nt) degradation signals called N-degrons, and to target these proteins for degradation by the 26S proteasome or autophagy. GID4, a subunit of the GID ubiquitin ligase, is the main recognition component of the proline (Pro)/N-degron pathway. GID4 targets proteins through their Nt-Pro residue or a Pro at position 2, in the presence of specific downstream sequence motifs. Here we show that human GID4 can also recognize hydrophobic Nt-residues other than Pro. One example is the sequence Nt-IGLW, bearing Nt-Ile. Nt-IGLW binds to wild-type human GID4 with aKdof 16 μM, whereas the otherwise identical Nt-Pro–bearing sequence PGLW binds to GID4 more tightly, with aKdof 1.9 μM. Despite this difference in affinities of GID4 for Nt-IGLW vs. Nt-PGLW, we found that the GID4-mediated Pro/N-degron pathway of the yeastSaccharomyces cerevisiaecan target an Nt-IGLW–bearing protein for rapid degradation. We solved crystal structures of human GID4 bound to a peptide bearing Nt-Ile or Nt-Val. We also altered specific residues of human GID4 and measured the affinities of resulting mutant GID4s for Nt-IGLW and Nt-PGLW, thereby determining relative contributions of specific GID4 residues to the GID4-mediated recognition of Nt-Pro vs. Nt-residues other than Pro. These and related results advance the understanding of targeting by the Pro/N-degron pathway and greatly expand the substrate recognition range of the GID ubiquitin ligase in both human and yeast cells.

scholarly journals Gid10 as an alternative N-recognin of the Pro/N-degron pathway

2019 ◽  
Vol 116 (32) ◽  
pp. 15914-15923 ◽  
Author(s):  
Artem Melnykov ◽  
Shun-Jia Chen ◽  
Alexander Varshavsky
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Carbon Source ◽  
Ubiquitin Ligase ◽  
Growth Conditions ◽  
26S Proteasome ◽  
Sequence Motifs ◽  
Yeast Protein ◽  
Substrate Specificities ◽  
Nonfermentable Carbon Source ◽  
A Subunit

In eukaryotes, N-degron pathways (formerly “N-end rule pathways”) comprise a set of proteolytic systems whose unifying feature is their ability to recognize proteins containing N-terminal degradation signals called N-degrons, thereby causing degradation of these proteins by the 26S proteasome or autophagy. Gid4, a subunit of the GID ubiquitin ligase in the yeast Saccharomyces cerevisiae, is the recognition component (N-recognin) of the GID-mediated Pro/N-degron pathway. Gid4 targets proteins by recognizing their N-terminal Pro residues or a Pro at position 2, in the presence of distinct adjoining sequence motifs. Under conditions of low or absent glucose, cells make it through gluconeogenesis. When S. cerevisiae grows on a nonfermentable carbon source, its gluconeogenic enzymes Fbp1, Icl1, Mdh2, and Pck1 are expressed and long-lived. Transition to a medium containing glucose inhibits the synthesis of these enzymes and induces their degradation by the Gid4-dependent Pro/N-degron pathway. While studying yeast Gid4, we identified a similar but uncharacterized yeast protein (YGR066C), which we named Gid10. A screen for N-terminal peptide sequences that can bind to Gid10 showed that substrate specificities of Gid10 and Gid4 overlap but are not identical. Gid10 is not expressed under usual (unstressful) growth conditions, but is induced upon starvation or osmotic stresses. Using protein binding analyses and degradation assays with substrates of GID, we show that Gid10 can function as a specific N-recognin of the Pro/N-degron pathway.

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 Screening for Modulators of Spermine Tolerance Identifies Sky1, the SR Protein Kinase of Saccharomyces cerevisiae, as a Regulator of Polyamine Transport and Ion Homeostasis

2001 ◽  
Vol 21 (1) ◽  
pp. 175-184 ◽  
Author(s):  
Omri Erez ◽  
Chaim Kahana
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Osmotic Shock ◽  
Ion Homeostasis ◽  
Inorganic Ions ◽  
Yeast Cells ◽  
Wild Type ◽  
Sr Protein ◽  
Polyamine Transport ◽  
Increased Sensitivity

ABSTRACT Although most cells are capable of transporting polyamines, the mechanism that regulates polyamine transport in eukaryotes is still largely unknown. Using a genetic screen for clones capable of restoring spermine sensitivity to spermine-tolerant mutants ofSaccharomyces cerevisiae, we have demonstrated that Sky1p, a recently identified SR protein kinase, is a key regulator of polyamine transport. Yeast cells deleted for SKY1 developed tolerance to toxic levels of spermine, while overexpression of Sky1p in wild-type cells increased their sensitivity to spermine. Expression of the wild-type Sky1p but not of a catalytically inactive mutant restored sensitivity to spermine. SKY1 disruption results in dramatically reduced uptake of spermine, spermidine, and putrescine. In addition to spermine tolerance, sky1Δ cells exhibit increased tolerance to lithium and sodium ions but somewhat increased sensitivity to osmotic shock. The observed halotolerance suggests potential regulatory interaction between the transport of polyamines and inorganic ions, as suggested in the case of the Ptk2p, a recently described regulator of polyamine transport. We demonstrate that these two kinases act in two different signaling pathways. While deletion or overexpression of SKY1 did not significantly affect Pma1p activity, the ability of overexpressed Sky1p, Ptk1p, and Ptk2p to increase sensitivity to LiCl depends on the integrity ofPPZ1 but not of ENA1.

Evolution of ethylene by Saccharomyces cerevisiae as influenced by the carbon source for growth and the presence of air

1978 ◽  
Vol 24 (6) ◽  
pp. 637-642 ◽  
Author(s):  
K. C. Thomas ◽  
Mary Spencer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Carbon Source ◽  
Ethylene Production ◽  
Atp Synthesis ◽  
Yeast Cells ◽  
Yeast Culture ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Absolute Requirement ◽  
Wild Type Yeast

Effects of the carbon source and oxygen on ethylene production by the yeast Saccharomyces cerevisiae have been studied. The amounts of ethylene evolved by the yeast culture were less than those detected in the blank (an equal volume of uninoculated medium), suggesting a net absorption of ethylene by the yeast cells. Addition of glucose to the lactate-grown yeast culture induced ethylene production. This glucose-induced stimulation of ethylene production was inhibited to a great extent by cycloheximide. Results suggested that the yeast cells in the presence of glucose synthesized an ethylene precursor and passed it into the medium. The conversion of this precursor to ethylene might be stimulated by oxygen. The fact that ethylene was produced by the yeast growing anaerobically and also by respiration-deficient mutants isolated from the wild-type yeast suggested that mitochondrial ATP synthesis was not an absolute requirement for ethylene biogenesis.

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

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.

scholarly journals Sml1 Inhibits the DNA Repair Activity of Rev1 in Saccharomyces cerevisiae during Oxidative Stress

2020 ◽  
Vol 86 (7) ◽  
Author(s):  
Rui Yao ◽  
Pei Zhou ◽  
Chengjin Wu ◽  
Liming Liu ◽  
Jing Wu
Keyword(s):  
Oxidative Stress ◽  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Survival Rate ◽  
Type Strain ◽  
Mutation Frequency ◽  
Wild Type Strain ◽  
Yeast Cells ◽  
Wild Type ◽  
Content Type

ABSTRACT In Saccharomyces cerevisiae, Y family DNA polymerase Rev1 is involved in the repair of DNA damage by translesion DNA synthesis (TLS). In the current study, to elucidate the role of Rev1 in oxidative stress-induced DNA damage in S. cerevisiae, REV1 was deleted and overexpressed; transcriptome analysis of these mutants along with the wild-type strain was performed to screen potential genes that could be associated with REV1 during response to DNA damage. When the yeast cells were treated with 2 mM H2O2, the deletion of REV1 resulted in a 1.5- and 2.8-fold decrease in the survival rate and mutation frequency, respectively, whereas overexpression of REV1 increased the survival rate and mutation frequency by 1.1- and 2.9-fold, respectively, compared to the survival rate and mutation frequency of the wild-type strain. Transcriptome and phenotypic analyses identified that Sml1 aggravated oxidative stress in the yeast cells by inhibiting the activity of Rev1. This inhibition was due to the physical interaction between the BRCA1 C terminus (BRCT) domain of Rev1 and amino acid residues 36 to 70 of Sml1; the cell survival rate and mutation frequency increased by 1.8- and 3.1-fold, respectively, when this interaction was blocked. We also found that Sml1 inhibited Rev1 phosphorylation under oxidative stress and that deletion of SML1 increased the phosphorylation of Rev1 by 46%, whereas overexpression of SML1 reduced phosphorylation of Rev1. Overall, these findings demonstrate that Sml1 could be a novel regulator that mediates Rev1 dephosphorylation to inhibit its activity during oxidative stress. IMPORTANCE Rev1 was critical for cell growth in S. cerevisiae, and the deletion of REV1 caused a severe growth defect in cells exposed to oxidative stress (2 mM H2O2). Furthermore, we found that Sml1 physically interacted with Rev1 and inhibited Rev1 phosphorylation, thereby inhibiting Rev1 DNA antioxidant activity. These findings indicate that Sml1 could be a novel regulator for Rev1 in response to DNA damage by oxidative stress.

scholarly journals Purification of profilin from Saccharomyces cerevisiae and analysis of profilin-deficient cells.

1990 ◽  
Vol 110 (1) ◽  
pp. 105-114 ◽  
Author(s):  
B K Haarer ◽  
S H Lillie ◽  
A E Adams ◽  
V Magdolen ◽  
W Bandlow ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Actin Polymerization ◽  
Yeast Cells ◽  
Predicted Amino Acid Sequence ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Ellipsoidal Shape ◽  
Hydrolysis Of

We have isolated profilin from yeast (Saccharomyces cerevisiae) and have microsequenced a portion of the protein to confirm its identity; the region microsequenced agrees with the predicted amino acid sequence from a profilin gene recently isolated from S. cerevisiae (Magdolen, V., U. Oechsner, G. Müller, and W. Bandlow. 1988. Mol. Cell. Biol. 8:5108-5115). Yeast profilin resembles profilins from other organisms in molecular mass and in the ability to bind to polyproline, retard the rate of actin polymerization, and inhibit hydrolysis of ATP by monomeric actin. Using strains that carry disruptions or deletions of the profilin gene, we have found that, under appropriate conditions, cells can survive without detectable profilin. Such cells grow slowly, are temperature sensitive, lose the normal ellipsoidal shape of yeast cells, often become multinucleate, and generally grow much larger than wild-type cells. In addition, these cells exhibit delocalized deposition of cell wall chitin and have dramatically altered actin distributions.

scholarly journals RSR1, a ras-like gene homologous to Krev-1 (smg21A/rap1A): role in the development of cell polarity and interactions with the Ras pathway in Saccharomyces cerevisiae.

1992 ◽  
Vol 12 (2) ◽  
pp. 758-766 ◽  
Author(s):  
R Ruggieri ◽  
A Bender ◽  
Y Matsui ◽  
S Powers ◽  
Y Takai ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Polarity ◽  
Copy Number ◽  
Mammalian Cells ◽  
Direct Interaction ◽  
Dominant Negative ◽  
Yeast Cells ◽  
Wild Type ◽  
Ras Gene ◽  
Ras Pathway

The Saccharomyces cerevisiae ras-like gene RSR1 is particularly closely related to the mammalian gene Krev-1 (also known as smg21A and rap1A). RSR1 was originally isolated as a multicopy suppressor of a cdc24 mutation, which causes an inability to bud or establish cell polarity. Deletion of RSR1 itself does not affect growth but causes a randomization of bud position. We have now constructed mutant alleles of RSR1 encoding proteins with substitutions of Val for Gly at position 12 (analogous to constitutively activated Ras proteins) or Asn for Lys at position 16 (analogous to a dominant-negative Ras protein). rsr1Val-12 could not restore a normal budding pattern to an rsr1 deletion strain but could suppress a cdc24 mutation when overexpressed. rsr1Asn-16 could randomize the budding pattern of a wild-type strain even in low copy number but was not lethal even in high copy number. These and other results suggest that Rsr1p functions only in bud site selection and not in subsequent events of polarity establishment and bud formation, that this function involves a cycling between GTP-bound and GDP-bound forms of the protein, and that the suppression of cdc24 involves direct interaction between Rsr1p[GTP] and Cdc24p. Functional homology between Rsr1p and Krev-1 p21 was suggested by the observations that expression of the latter protein in yeast cells could both suppress a cdc24 mutation and randomize the budding pattern of wild-type cells. As Krev-1 overexpression can suppress ras-induced transformation of mammalian cells, we looked for effects of RSR1 on the S. cerevisiae Ras pathway. Although no suppression of the activated RAS2Val-19 allele was observed, overexpression of rsr1Val-12 suppressed the lethality of strains lacking RAS gene function, apparently through a direct activation of adenyl cyclase. This interaction of Rsr1p with the effector of Ras in S. cerevisiae suggests that Krev-1 may revert ras-induced transformation of mammalian cells by affecting the interaction of ras p21 with its effector.

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.

scholarly journals A stalled retrotranslocation complex reveals physical linkage between substrate recognition and proteasomal degradation during ER-associated degradation

2013 ◽  
Vol 24 (11) ◽  
pp. 1765-1775 ◽  
Author(s):  
Kunio Nakatsukasa ◽  
Jeffrey L. Brodsky ◽  
Takumi Kamura
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Ubiquitin Ligase ◽  
Substrate Recognition ◽  
Proteasomal Degradation ◽  
26S Proteasome ◽  
Endoplasmic Reticulum Associated Degradation ◽  
Ubiquitin Ligase Complex ◽  
Physical Linkage ◽  
Tightly Coupled

During endoplasmic reticulum–associated degradation (ERAD), misfolded lumenal and membrane proteins in the ER are recognized by the transmembrane Hrd1 ubiquitin ligase complex and retrotranslocated to the cytosol for ubiquitination and degradation. Although substrates are believed to be delivered to the proteasome only after the ATPase Cdc48p/p97 acts, there is limited knowledge about how the Hrd1 complex coordinates with Cdc48p/p97 and the proteasome to orchestrate substrate recognition and degradation. Here we provide evidence that inactivation of Cdc48p/p97 stalls retrotranslocation and triggers formation of a complex that contains the 26S proteasome, Cdc48p/p97, ubiquitinated substrates, select components of the Hrd1 complex, and the lumenal recognition factor, Yos9p. We propose that the actions of Cdc48p/p97 and the proteasome are tightly coupled during ERAD. Our data also support a model in which the Hrd1 complex links substrate recognition and degradation on opposite sides of the ER membrane.

Sign in / Sign up

Export Citation Format

Share Document