scholarly journals Timescale of degradation-driven protein level fluctuation in the yeast Saccharomyces cerevisiae

2021 ◽  
Author(s):  
Bahareh Mahrou ◽  
Azady Pirhanov ◽  
Moluk Hadi Alijanvand ◽  
Yong Ku Cho ◽  
Yong-Jun Shin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Degradation ◽  
Protein Level ◽  
Transcriptional Control ◽  
Degradation Rate ◽  
Modulation Frequency ◽  
Cellular Protein ◽  
Yeast Saccharomyces Cerevisiae ◽  
Protein Levels ◽  
Protein Degradation Rate

Generating robust, predictable perturbations in cellular protein levels will advance our understanding of protein function and enable control of physiological outcomes in biotechnology applications. Previous studies have shown that controlling RNA transcription achieves perturbations in protein levels over a timescale of several hours. Here, we demonstrate the potential for harnessing the protein degradation machinery to achieve robust, rapid control of a specific protein level in the yeast Saccharomyces cerevisiae. Using a light-driven protein degradation machinery and red fluorescent proteins as reporters, we show that under constant transcriptional induction, repeated triangular fluctuations in protein levels can be generated by controlling the protein degradation rate. Consistent with previous results using transcriptional control, we observed a continuous decrease in the magnitude of fluctuations as the modulation frequency increased, indicating low-pass filtering of input perturbation. However, compared to hour-scale fluctuations observed using transcriptional control, modulating the protein degradation rate enabled five to ten minute-scale fluctuations. Our study demonstrates the potential for repeated control of protein levels by controlling protein degradation rate, at timescales much shorter than that achieved by transcriptional control.

scholarly journals Absence of heme at the catalytic site of heme oxygenase-2 triggers its lysosomal degradation

2020 ◽  
Author(s):  
Liu Liu ◽  
Arti B. Dumbrepatil ◽  
Angela S. Fleischhacker ◽  
E. Neil G. Marsh ◽  
Stephen W. Ragsdale
Keyword(s):  
Heme Oxygenase ◽  
Protein Level ◽  
Translational Regulation ◽  
Transcriptional Control ◽  
Degradation Rate ◽  
Cellular Protein ◽  
Catalytic Site ◽  
Heme Binding ◽  
Low Protein ◽  
Pathophysiological Role

ABSTRACTHeme oxygenase-2 (HO2) and −1 (HO1) catalyze heme degradation to biliverdin, CO, and iron, forming an essential link in the heme metabolism network. Tight regulation of the cellular levels and catalytic activities of HO1 and HO2 is important for maintaining heme homeostasis. While transcriptional control of HO1 expression has been well-studied, how the cellular levels and activity of HO2 are regulated remains unclear. Here, the mechanism of post-translational regulation of cellular HO2 level by heme is elucidated. Under heme deficient conditions, HO2 is destabilized and targeted for degradation. In HO2, three heme binding sites are potential targets of heme-dependent regulation: one at its catalytic site; the others at its two heme regulatory motifs (HRMs). We report that, in contrast to other HRM-containing proteins, the cellular protein level and degradation rate of HO2 are independent of heme binding to the HRMs. Rather, under heme deficiency, loss of heme binding to the catalytic site destabilizes HO2. Consistently, a HO2 catalytic site variant that is unable to bind heme exhibits a constant low protein level and an enhanced protein degradation rate compared to the wild-type HO2. However, cellular heme overload does not affect HO2 stability. Finally, HO2 is degraded by the lysosome through chaperone-mediated autophagy, distinct from other HRM-containing proteins and HO1, which are degraded by the proteasome. These results reveal a novel aspect of HO2 regulation and deepen our understanding of HO2’s role in maintaining heme homeostasis, paving the way for future investigation into HO2’s pathophysiological role in heme deficiency response.

scholarly journals Cloning and characterization of four SIR genes of Saccharomyces cerevisiae.

1986 ◽  
Vol 6 (2) ◽  
pp. 688-702 ◽  
Author(s):  
J M Ivy ◽  
A J Klar ◽  
J B Hicks
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mating Type ◽  
Blot Analysis ◽  
Transcriptional Control ◽  
Genomic Library ◽  
Yeast Saccharomyces Cerevisiae ◽  
Mating Type Genes ◽  
Rna Blots

Mating type in the yeast Saccharomyces cerevisiae is determined by the MAT (a or alpha) locus. HML and HMR, which usually contain copies of alpha and a mating type information, respectively, serve as donors in mating type interconversion and are under negative transcriptional control. Four trans-acting SIR (silent information regulator) loci are required for repression of transcription. A defect in any SIR gene results in expression of both HML and HMR. The four SIR genes were isolated from a genomic library by complementation of sir mutations in vivo. DNA blot analysis suggests that the four SIR genes share no sequence homology. RNA blots indicate that SIR2, SIR3, and SIR4 each encode one transcript and that SIR1 encodes two transcripts. Null mutations, made by replacement of the normal genomic allele with deletion-insertion mutations created in the cloned SIR genes, have a Sir- phenotype and are viable. Using the cloned genes, we showed that SIR3 at a high copy number is able to suppress mutations of SIR4. RNA blot analysis suggests that this suppression is not due to transcriptional regulation of SIR3 by SIR4; nor does any SIR4 gene transcriptionally regulate another SIR gene. Interestingly, a truncated SIR4 gene disrupts regulation of the silent mating type loci. We propose that interaction of at least the SIR3 and SIR4 gene products is involved in regulation of the silent mating type genes.

Impact of Weighing Procedures and Variation in Protein Degradation Rate on Measured Performance of Growing Lambs and Cattle

1983 ◽  
Vol 57 (5) ◽  
pp. 1276-1285 ◽  
Author(s):  
Rick Stock ◽  
Terry Klopfenstein ◽  
Dennis Brink ◽  
Steve Lowry ◽  
Dave Rock ◽  
...  
Keyword(s):  
Protein Degradation ◽  
Degradation Rate ◽  
Protein Degradation Rate

scholarly journals Heme oxygenase-2 is post-translationally regulated by heme occupancy in the catalytic site

2020 ◽  
Vol 295 (50) ◽  
pp. 17227-17240 ◽  
Author(s):  
Liu Liu ◽  
Arti B. Dumbrepatil ◽  
Angela S. Fleischhacker ◽  
E. Neil G. Marsh ◽  
Stephen W. Ragsdale
Keyword(s):  
Heme Oxygenase ◽  
Protein Level ◽  
Translational Regulation ◽  
Degradation Rate ◽  
Cellular Protein ◽  
Catalytic Site ◽  
Heme Binding ◽  
Direct Role ◽  
Low Protein ◽  
Pathophysiological Role

Heme oxygenase-2 (HO2) and -1 (HO1) catalyze heme degradation to biliverdin, CO, and iron, forming an essential link in the heme metabolism network. Tight regulation of the cellular levels and catalytic activities of HO1 and HO2 is important for maintaining heme homeostasis. HO1 expression is transcriptionally regulated; however, HO2 expression is constitutive. How the cellular levels and activity of HO2 are regulated remains unclear. Here, we elucidate the mechanism of post-translational regulation of cellular HO2 levels by heme. We find that, under heme-deficient conditions, HO2 is destabilized and targeted for degradation, suggesting that heme plays a direct role in HO2 regulation. HO2 has three heme binding sites: one at its catalytic site and the others at its two heme regulatory motifs (HRMs). We report that, in contrast to other HRM-containing proteins, the cellular protein level and degradation rate of HO2 are independent of heme binding to the HRMs. Rather, under heme deficiency, loss of heme binding to the catalytic site destabilizes HO2. Consistently, an HO2 catalytic site variant that is unable to bind heme exhibits a constant low protein level and an enhanced protein degradation rate compared with the WT HO2. Finally, HO2 is degraded by the lysosome through chaperone-mediated autophagy, distinct from other HRM-containing proteins and HO1, which are degraded by the proteasome. These results reveal a novel aspect of HO2 regulation and deepen our understanding of HO2's role in maintaining heme homeostasis, paving the way for future investigation into HO2's pathophysiological role in heme deficiency response.

scholarly journals TFG/TAF30/ANC1, a component of the yeast SWI/SNF complex that is similar to the leukemogenic proteins ENL and AF-9.

1996 ◽  
Vol 16 (7) ◽  
pp. 3308-3316 ◽  
Author(s):  
B R Cairns ◽  
N L Henry ◽  
R D Kornberg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Acute Leukemia ◽  
Rna Polymerase Ii ◽  
Protein Interaction ◽  
Transcriptional Control ◽  
Gene Products ◽  
Yeast Saccharomyces Cerevisiae ◽  
Genetic Studies ◽  
Biochemical Studies ◽  
Transcription Complexes

The SWI1/ADR6, SWI2/SNF2, SWI3, SNF5, and SNF6 gene products are all required for proper transcriptional control of many genes in the yeast Saccharomyces cerevisiae. Genetic studies indicated that these gene products might form a multiprotein SWI/SNF complex important for chromatin transitions preceding transcription from RNA polymerase II promoters. Biochemical studies identified a SWI/SNF complex containing these and at least six additional polypeptides. Here we show that the 29-kDa component of the SWI/SNF complex is identical to TFG3/TAF30/ANC1. Thus, a component of the SWI/SNF complex is also a member of the TFIIF and TFIID transcription complexes. TFG3 interacted with the SNF5 component of the SWI/SNF complex in protein interaction blots. TFG3 is significantly similar to ENL and AF-9, two proteins implicated in human acute leukemia. These results suggest that ENL and AF-9 proteins interact with the SNF5 component of the human SWI/SNF complex and raise the possibility that the SWI/SNF complex is involved in acute leukemia.

Overexpression of Akt2 in Oral Cancer Cells is Due To Reduced Protein Degradation Rate

2015 ◽  
Vol 119 (3) ◽  
pp. e108
Author(s):  
Anak Iamaroon ◽  
Supansa Pata ◽  
Prakasit Archewa ◽  
Chayarop Supanachart ◽  
Sutthichai Krisanaprakornkit
Keyword(s):  
Oral Cancer ◽  
Protein Degradation ◽  
Cancer Cells ◽  
Degradation Rate ◽  
Oral Cancer Cells ◽  
Protein Degradation Rate

scholarly journals Effect of heterologous expression of acyl-CoA-binding protein on acyl-CoA level and composition in yeast

1993 ◽  
Vol 290 (2) ◽  
pp. 369-374 ◽  
Author(s):  
S Mandrup ◽  
R Jepsen ◽  
H Skøtt ◽  
J Rosendal ◽  
P Højrup ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Growth Rate ◽  
Heterologous Expression ◽  
Binding Protein ◽  
Cellular Protein ◽  
Serine Residue ◽  
Yeast Saccharomyces Cerevisiae ◽  
Total Cellular Protein ◽  
Gal1 Promoter

We have expressed a bovine synthetic acyl-CoA-binding protein (ACBP) gene in yeast (Saccharomyces cerevisiae) under the control of the GAL1 promoter. The heterologously expressed bovine ACBP constituted up to 6.4% of total cellular protein and the processing was identical with that of native bovine ACBP, i.e. the initiating methionine was removed and the following serine residue was N-acetylated. The expression of this protein did not affect the growth rate of the cells. Determination of the yeast acyl-CoA pool size showed a close positive correlation between the ACBP content of the cells and the size of the acyl-CoA pool. Thus ACBP can act as an intracellular acyl-CoA pool former. Possible physiological functions of ACBP in cells are discussed.

In vitro ruminal undegradable proteins of alfalfa cultivars

2000 ◽  
Vol 80 (2) ◽  
pp. 315-325 ◽  
Author(s):  
G. F. Tremblay ◽  
R. Michaud ◽  
G. Bélanger ◽  
K. B. McRae ◽  
H. V. Petit
Keyword(s):  
Protein Degradation ◽  
Dry Matter ◽  
Degradation Rate ◽  
Weight Ratio ◽  
Principal Component ◽  
Neutral Detergent Fiber ◽  
Cultivar Differences ◽  
Dry Matter Digestibility ◽  
Protein Degradation Rate

The quality of alfalfa would be greatly improved by an increase in its ruminal undegradable protein (RUP) concentration. Protein degradation rate (PDR), in vitro dry matter digestibility (IVDMD), leaf weight ratio (LWR), dry matter yield (DMY), total nitrogen (TN), in vitro RUP (expressed on both TN, RUP-TN, and dry matter basis, RUP-DM), acid detergent fiber (ADF), and neutral detergent fiber (NDF) concentrations were determined in 27 alfalfa cultivars. Cultivars were seeded in triplicate on 2 consecutive years and evaluated during the 2 subsequent production years with two harvests per year. Protein degradation rate and RUP-TN were determined using a ruminal inhibitor in vitro system. Data were averaged for spring growth, summer regrowth, and both harvests across 2 production years. Each of the three data sets was analyzed by ANOVA followed by a principal component analysis (PCA) on the ANOVA means. For the four-harvest data, cultivar differences were highly significant (P < 0.001) for all variates except for PDR (P = 0.07) and RUP-TN concentration (P = 0.10). The first PCA axis was largely defined positively by RUP-DM, IVDMD, TN, LWR, and RUP-TN, but negatively with ADF, NDF, PDR, and DMY. The second PCA axis defined a contrast between PDR versus RUP-TN, DMY, ADF, and NDF. Five cultivars were distinctive with high or low PCA scores in all three PCA. Rangelander and Heinrichs, along with Ultra, had low PDR; the first two cultivars had low DMY whereas Ultra was a medium-yielding cultivar. In contrast, Algonquin and Oneida VR had high PDR and medium DMY. While the first principal component (PC) indicated a general trend that low PDR and high RUP were associated with low-yielding cultivars, the second PC identified specific cultivars with both low PDR and high DMY. Therefore, selection for low PDR and high DMY is feasible. Key words: ruminal protein escape, dry matter digestibility, alfalfa

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