scholarly journals Asi1 regulates the distribution of proteins at the inner nuclear membrane in Saccharomyces cerevisiae

2018 ◽  
Author(s):  
Christine J. Smoyer ◽  
Sarah E. Smith ◽  
Scott McCroskey ◽  
Jay R. Unruh ◽  
Sue L. Jaspersen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Membrane ◽  
Protein Turnover ◽  
Protein Quality ◽  
E3 Ligase ◽  
Protein Composition ◽  
Degradation Pathways ◽  
Wild Type ◽  
Inner Nuclear Membrane ◽  
Protein Levels

AbstractInner nuclear membrane (INM) protein composition regulates nuclear function, affecting processes such as gene expression, chromosome organization, nuclear shape and stability. Mechanisms that drive changes in the INM proteome are poorly understood in part because it is difficult to definitively assay INM composition rigorously and systematically. Using a split-GFP complementation system to detect INM access, we examined the distribution of all C-terminally tagged Saccharomyces cerevisiae membrane proteins in wild-type cells and in mutants affecting protein quality control pathways, such as INM-associated degradation (INMAD), ER-associated degradation (ERAD) and vacuolar proteolysis. Deletion of the E3 ligase Asi1 had the most pronounced effect on the INM compared to mutants in vacuolar or ER-associated degradation pathways, consistent with a role for Asi1 in the INMAD pathway. Our data suggests that Asi1 not only removes mis-targeted proteins at the INM, but it also controls the levels and distribution of native INM components, such as the membrane nucleoporin Pom33. Interestingly, loss of Asi1 does not affect Pom33 protein levels but instead alters Pom33 distribution in the NE through Pom33 ubiquitination, which drives INM redistribution. Taken together, our data demonstrate that the Asi1 E3 ligase has a novel function in INM protein regulation in addition to protein turnover.

scholarly journals Distribution of Proteins at the Inner Nuclear Membrane Is Regulated by the Asi1 E3 Ligase in Saccharomyces cerevisiae

Genetics ◽  
2019 ◽  
Vol 211 (4) ◽  
pp. 1269-1282 ◽  
Author(s):  
Christine J. Smoyer ◽  
Sarah E. Smith ◽  
Jennifer M. Gardner ◽  
Scott McCroskey ◽  
Jay R. Unruh ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Membrane ◽  
E3 Ligase ◽  
Inner Nuclear Membrane

scholarly journals A distinct inner nuclear membrane proteome in Saccharomyces cerevisiae gametes

2021 ◽  
Author(s):  
Shary N Shelton ◽  
Sarah E Smith ◽  
Jay R Unruh ◽  
Sue L Jaspersen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Membrane ◽  
De Novo ◽  
Protein Composition ◽  
Parental Cell ◽  
Permeability Barrier ◽  
Developmentally Regulated ◽  
Inner Nuclear Membrane ◽  
Expression Of Genes ◽  
Mitotic Cells

Abstract The inner nuclear membrane (INM) proteome regulates gene expression, chromatin organization, and nuclear transport; however, it is poorly understood how changes in INM protein composition contribute to developmentally regulated processes, such as gametogenesis. We conducted a screen to determine how the INM proteome differs between mitotic cells and gametes. In addition, we used a strategy that allowed us to determine if spores synthesize their INM proteins de novo, rather than inheriting their INM proteins from the parental cell. This screen used a split-GFP complementation system, where we were able to compare the distribution of all C-terminally tagged transmembrane proteins in Saccharomyces cerevisiae in gametes to that of mitotic cells. Gametes contain a distinct INM proteome needed to complete gamete formation, including expression of genes linked to cell wall biosynthesis, lipid biosynthetic and metabolic pathways, protein degradation, and unknown functions. Based on the inheritance pattern, INM components are made de novo in the gametes. Whereas mitotic cells show a strong preference for proteins with small extraluminal domains, gametes do not exhibit this size preference likely due to the changes in the nuclear permeability barrier during gametogenesis. Taken together, our data provide evidence for INM changes during gametogenesis and shed light on mechanisms used to shape the INM proteome of spores.

scholarly journals A distinct inner nuclear membrane proteome in Saccharomyces cerevisiae gametes

2021 ◽  
Author(s):  
Shary N Shelton ◽  
Sarah E. Smith ◽  
Jay R. Unruh ◽  
Sue L. Jaspersen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Membrane ◽  
De Novo ◽  
Protein Composition ◽  
Permeability Barrier ◽  
Developmentally Regulated ◽  
Inner Nuclear Membrane ◽  
Expression Of Genes ◽  
Size Preference ◽  
Mitotic Cells

The inner nuclear membrane (INM) proteome regulates gene expression, chromatin organization, and nuclear transport, however, it is poorly understood how changes in INM protein composition contribute to developmentally regulated processes, such as gametogenesis. Using a split-GFP complementation system, we compared the distribution of all C-terminally tagged transmembrane proteins in Saccharomyces cerevisiae in gametes to that of mitotic cells. Gametes contain a distinct INM proteome needed to complete gamete formation, including expression of genes linked to cell wall biosynthesis, lipid biosynthetic and metabolic pathways, protein degradation and unknown functions. Based on the inheritance pattern, INM components are made de novo in the gametes. Whereas mitotic cells show a strong preference for proteins with small extraluminal domains, gametes do not exhibit this size preference likely due to the changes in the nuclear permeability barrier during gametogenesis.

scholarly journals Influences of Histone Stoichiometry on the Target Site Preference of Retrotransposons Ty1 and Ty2 in Saccharomyces cerevisiae

Genetics ◽  
1996 ◽  
Vol 142 (3) ◽  
pp. 761-776 ◽  
Author(s):  
Lori A Rinckel ◽  
David J Garfinkel
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Promoter Region ◽  
Target Site ◽  
Site Preference ◽  
Wild Type ◽  
Histone Proteins ◽  
Protein Levels ◽  
In The Wild ◽  
Type Strains ◽  
Orientation Bias

Abstract In Saccharomyces cerevisiae, the target site specificity of the retrotransposon Ty1 appears to involve the Ty integration complex recognizing chromatin structures. To determine whether changes in chromatin structure affect Ty1 and Ty2 target site preference, we analyzed Ty transposition at the CAN1 locus in mutants containing altered levels of histone proteins. A Δhta1-htb1 mutant with decreased levels of H2A and H2B histone proteins showed a pattern of Ty1 and Ty2 insertions at CAN1 that was significantly different from that of both the wild-type and a Δhta2-htb2 mutant, which does not have altered histone protein levels. Altered levels of H2A and H2B proteins disrupted a dramatic orientation bias in the CAN1 promoter region. In the wild-type strains, few Ty1 and Ty2 insertions in the promoter region were oriented opposite to the direction of CAN1 transcription. In the Δhta1-htb1 background, however, numerous Ty1 and Ty2 insertions were in the opposite orientation clustered within the TATA region. This altered insertion pattern does not appear to be due to a bias caused by selecting canavanine resistant isolates in the different HTA1-HTB1 backgrounds. Our results suggest that reduced levels of histone proteins alter Ty target site preference and disrupt an asymmetric Ty insertion pattern.

scholarly journals Btn2 is involved in the clearance of denatured proteins caused by severe ethanol stress in Saccharomyces cerevisiae

2019 ◽  
Author(s):  
Sae Kato ◽  
Masashi Yoshida ◽  
Shingo Izawa
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Protein Quality ◽  
Protein Denaturation ◽  
Recovery Process ◽  
Ubiquitin Proteasome System ◽  
Yeast Cells ◽  
Ethanol Stress ◽  
Protein Levels ◽  
Denatured Proteins

Abstract Saccharomyces cerevisiae shows similar responses to heat shock and ethanol stress. Cells treated with severe ethanol stress activate the transcription of HSP genes and cause the aggregation of Hsp104-GFP, implying that severe ethanol stress as well as heat shock causes the accumulation of denatured proteins in yeast cells. However, there is currently no concrete evidence to show that severe ethanol stress causes protein denaturation in living yeast cells. In the present study, we investigated whether severe ethanol stress causes protein denaturation, and confirmed that a treatment with 10% (v/v) ethanol stress resulted in the accumulation of insoluble proteins and ubiquitinated proteins in yeast cells. We also found that increased denatured protein levels were efficiently reduced by the ubiquitin-proteasome system after the elimination of ethanol. Since our previous findings demonstrated that the expression of Btn2 was induced by severe ethanol stress, we herein examined the importance of Btn2 in protein quality control in cells treated with severe ethanol stress. btn2∆ cells showed a significant delay in the clearance of denatured proteins during the recovery process. These results provide further insights into the effects of severe ethanol on yeast proteostasis and the contribution of Btn2 to the efficient clearance of denatured proteins.

scholarly journals Crosstalk and ultrasensitivity in protein degradation pathways

2020 ◽  
Vol 16 (12) ◽  
pp. e1008492
Author(s):  
Abhishek Mallela ◽  
Maulik K. Nariya ◽  
Eric J. Deeds
Keyword(s):  
Protein Turnover ◽  
E3 Ligase ◽  
Biochemical Networks ◽  
Protein Homeostasis ◽  
Polyubiquitin Chain ◽  
Post Translational Modification ◽  
E3 Ligases ◽  
Protein Levels ◽  
Synthesis And Degradation

Protein turnover is vital to cellular homeostasis. Many proteins are degraded efficiently only after they have been post-translationally “tagged” with a polyubiquitin chain. Ubiquitylation is a form of Post-Translational Modification (PTM): addition of a ubiquitin to the chain is catalyzed by E3 ligases, and removal of ubiquitin is catalyzed by a De-UBiquitylating enzyme (DUB). Nearly four decades ago, Goldbeter and Koshland discovered that reversible PTM cycles function like on-off switches when the substrates are at saturating concentrations. Although this finding has had profound implications for the understanding of switch-like behavior in biochemical networks, the general behavior of PTM cycles subject to synthesis and degradation has not been studied. Using a mathematical modeling approach, we found that simply introducing protein turnover to a standard modification cycle has profound effects, including significantly reducing the switch-like nature of the response. Our findings suggest that many classic results on PTM cycles may not hold in vivo where protein turnover is ubiquitous. We also found that proteins sharing an E3 ligase can have closely related changes in their expression levels. These results imply that it may be difficult to interpret experimental results obtained from either overexpressing or knocking down protein levels, since changes in protein expression can be coupled via E3 ligase crosstalk. Understanding crosstalk and competition for E3 ligases will be key in ultimately developing a global picture of protein homeostasis.

scholarly journals The ClpP Peptidase Is the Major Determinant of Bulk Protein Turnover in Bacillus subtilis

2004 ◽  
Vol 186 (17) ◽  
pp. 5856-5864 ◽  
Author(s):  
Holger Kock ◽  
Ulf Gerth ◽  
Michael Hecker
Keyword(s):  
Bacillus Subtilis ◽  
Protein Turnover ◽  
Protein Quality ◽  
Regulatory Proteins ◽  
Wild Type ◽  
Intracellular Protein ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases ◽  
Degradation Rates ◽  
The One

ABSTRACT Measurements of overall protein degradation rates in wild-type and clpP mutant Bacillus subtilis cells revealed that stress- or starvation-induced bulk protein turnover depends virtually exclusively on the ClpP peptidase. ClpP is also essential for intracellular protein quality control, and in its absence newly synthesized proteins were highly prone to aggregation even at 37°C. Proteomic comparisons between the wild type and a ΔclpP mutant showed that the absence of ClpP leads to severe perturbations of “normal” physiology, complicating the detection of ClpP substrates. A pulse-chase two-dimensional gel approach was therefore used to compare wild-type and clpP mutant cultures that had been radiolabeled in mid-exponential phase, by quantifying changes in relative spot intensities with time. The results showed that overall proteolysis is biased toward proteins with vegetative functions which are no longer required (or are required at lower levels) in the nongrowing state. The identified substrate candidates for ClpP-dependent degradation include metabolic enzymes and aminoacyl-tRNA synthetases. Some substrate candidates catalyze the first committed step of certain biosynthetic pathways. Our data suggest that ClpP-dependent proteolysis spans a broad physiological spectrum, with regulatory processing of key metabolic components and regulatory proteins on the one side and general bulk protein breakdown at the transition from growing to nongrowing phases on the other.

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