scholarly journals Phototropin Interactions with SUMO Proteins

2021 ◽  
Author(s):  
Justyna Łabuz ◽  
Olga Sztatelman ◽  
Dominika Jagiełło-Flasińska ◽  
Paweł Hermanowicz ◽  
Aneta Bażant ◽  
...  
Keyword(s):  
Blue Light ◽  
Covalent Modification ◽  
Mass Spectrometry Analysis ◽  
Protein Abundance ◽  
Growth Responses ◽  
Yeast Two Hybrid ◽  
Spectrometry Analysis ◽  
Mature Leaves ◽  
The Stability ◽  
Two Hybrid

Abstract The disruption of the sumoylation pathway affects processes controlled by the two phototropins (phots) of Arabidopsis thaliana, phot1 and phot2. Phots, plant UVA/blue light photoreceptors, regulate growth responses and fast movements aimed at optimizing photosynthesis, such as phototropism, chloroplast relocations and stomatal opening. Sumoylation is a posttranslational modification, consisting of the addition of a SUMO (SMALL UBIQUITIN-RELATED MODIFIER) protein to a lysine residue in the target protein. In addition to affecting the stability of proteins, it regulates their activity, interactions and subcellular localization. We examined physiological responses controlled by phots, phototropism and chloroplast movements, in sumoylation pathway mutants. Chloroplast accumulation in response to both continuous and pulse light was enhanced in the E3 ligase siz1 mutant, in a manner dependent on phot2. A significant decrease in phot2 protein abundance was observed in this mutant after blue light treatment both in seedlings and mature leaves. Using plant transient expression and yeast two-hybrid assays, we found that phots interacted with SUMO proteins mainly through their N-terminal parts, which contain the photosensory LOV domains. The covalent modification in phots by SUMO was verified using an Arabidopsis sumoylation system reconstituted in bacteria followed by the mass spectrometry analysis. Lys 297 was identified as the main target of SUMO3 in the phot2 molecule. Finally, sumoylation of phot2 was detected in Arabidopsis mature leaves upon light or heat stress treatment.

scholarly journals Shared Components of the FRQ-Less Oscillator and TOR Pathway Maintain Rhythmicity in Neurospora

2021 ◽  
pp. 074873042199994
Author(s):  
Rosa Eskandari ◽  
Lalanthi Ratnayake ◽  
Patricia L. Lakin-Thomas
Keyword(s):  
Circadian Rhythms ◽  
Clock Genes ◽  
Nutrient Sensing ◽  
Mass Spectrometry Analysis ◽  
Growth Responses ◽  
Tor Pathway ◽  
Spectrometry Analysis ◽  
Binding Partner ◽  
Binding Partners ◽  
Free Running

Molecular models for the endogenous oscillators that drive circadian rhythms in eukaryotes center on rhythmic transcription/translation of a small number of “clock genes.” Although substantial evidence supports the concept that negative and positive transcription/translation feedback loops (TTFLs) are responsible for regulating the expression of these clock genes, certain rhythms in the filamentous fungus Neurospora crassa continue even when clock genes ( frq, wc-1, and wc-2) are not rhythmically expressed. Identification of the rhythmic processes operating outside of the TTFL has been a major unresolved area in circadian biology. Our lab previously identified a mutation ( vta) that abolishes FRQ-less rhythmicity of the conidiation rhythm and also affects rhythmicity when FRQ is functional. Further studies identified the vta gene product as a component of the TOR (Target of Rapamycin) nutrient-sensing pathway that is conserved in eukaryotes. We now report the discovery of TOR pathway components including GTR2 (homologous to the yeast protein Gtr2, and RAG C/D in mammals) as binding partners of VTA through co-immunoprecipitation (IP) and mass spectrometry analysis using a VTA-FLAG strain. Reciprocal IP with GTR2-FLAG found VTA as a binding partner. A Δ gtr2 strain was deficient in growth responses to amino acids. Free-running conidiation rhythms in a FRQ-less strain were abolished in Δ gtr2. Entrainment of a FRQ-less strain to cycles of heat pulses demonstrated that Δ gtr2 is defective in entrainment. In all of these assays, Δ gtr2 is similar to Δ vta. In addition, expression of GTR2 protein was found to be rhythmic across two circadian cycles, and functional VTA was required for GTR2 rhythmicity. FRQ protein exhibited the expected rhythm in the presence of GTR2 but the rhythmic level of FRQ dampened in the absence of GTR2. These results establish association of VTA with GTR2, and their role in maintaining functional circadian rhythms through the TOR pathway.

scholarly journals Structural Basis of GLUT1 Inhibition by Cytoplasmic ATP

2007 ◽  
Vol 130 (2) ◽  
pp. 157-168 ◽  
Author(s):  
David M. Blodgett ◽  
Julie K. De Zutter ◽  
Kara B. Levine ◽  
Pusha Karim ◽  
Anthony Carruthers
Keyword(s):  
Glucose Transport ◽  
Conformational Changes ◽  
Covalent Modification ◽  
Antibody Binding ◽  
Mass Spectrometry Analysis ◽  
Structural Basis ◽  
Ionization Mass ◽  
Spectrometry Analysis ◽  
C Terminus ◽  
Lysine Residues

Cytoplasmic ATP inhibits human erythrocyte glucose transport protein (GLUT1)–mediated glucose transport in human red blood cells by reducing net glucose transport but not exchange glucose transport (Cloherty, E.K., D.L. Diamond, K.S. Heard, and A. Carruthers. 1996. Biochemistry. 35:13231–13239). We investigated the mechanism of ATP regulation of GLUT1 by identifying GLUT1 domains that undergo significant conformational change upon GLUT1–ATP interaction. ATP (but not GTP) protects GLUT1 against tryptic digestion. Immunoblot analysis indicates that ATP protection extends across multiple GLUT1 domains. Peptide-directed antibody binding to full-length GLUT1 is reduced by ATP at two specific locations: exofacial loop 7–8 and the cytoplasmic C terminus. C-terminal antibody binding to wild-type GLUT1 expressed in HEK cells is inhibited by ATP but binding of the same antibody to a GLUT1–GLUT4 chimera in which loop 6–7 of GLUT1 is substituted with loop 6–7 of GLUT4 is unaffected. ATP reduces GLUT1 lysine covalent modification by sulfo-NHS-LC-biotin by 40%. AMP is without effect on lysine accessibility but antagonizes ATP inhibition of lysine modification. Tandem electrospray ionization mass spectrometry analysis indicates that ATP reduces covalent modification of lysine residues 245, 255, 256, and 477, whereas labeling at lysine residues 225, 229, and 230 is unchanged. Exogenous, intracellular GLUT1 C-terminal peptide mimics ATP modulation of transport whereas C-terminal peptide-directed IgGs inhibit ATP modulation of glucose transport. These findings suggest that transport regulation involves ATP-dependent conformational changes in (or interactions between) the GLUT1 C terminus and the C-terminal half of GLUT1 cytoplasmic loop 6–7.

E3 ubiquitin-protein ligases in rat kidney collecting duct: response to vasopressin stimulation and withdrawal

2011 ◽  
Vol 301 (4) ◽  
pp. F883-F896 ◽  
Author(s):  
Yu-Jung Lee ◽  
Jeong-Eun Lee ◽  
Hyo-Jung Choi ◽  
Jung-Suk Lim ◽  
Hyun Jun Jung ◽  
...  
Keyword(s):  
Transcriptome Analysis ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Urine Osmolality ◽  
Urine Concentration ◽  
Mass Spectrometry Analysis ◽  
Protein Abundance ◽  
Sprague Dawley ◽  
Spectrometry Analysis ◽  
Protein Ligases

The E3 ubiquitin (Ub)-protein ligases (E3s) play a role as regulators of protein trafficking and degradation. We aimed to integrate the profile of E3s in rat kidney and examine the changes in protein abundance of the selected E3s in response to 1-deamino-8-d-arginine vasopressin (dDAVP) stimulation/withdrawal. Sprague-Dawley rats were infused with vehicle ( n = 13), dDAVP for 5 days ( n = 13), or dDAVP was withdrawn for periods (15 min, 30 min, 1, 3, 6, 12, or 24 h) after 5-day infusion ( n = 46). Total RNA was isolated from the inner medulla (IM) for transcriptome analysis. Plasma membrane (PM)- or intracellular vesicle (ICV)-enriched fractions of whole kidney were immunoisolated for liquid chromatography-tandem mass spectrometry analysis. dDAVP infusion for 5 days (D5d) significantly increased urine osmolality, which was maintained during 3-h withdrawal of dDAVP after 5-day infusion (D5d-3h). Consistent with this, aquaporin-2 (AQP2) expression in the PM fractions of D5d and D5d-3h increased, whereas AQP2 expression in the ICV fractions of D5d-3h was further increased, indicating internalization of AQP2. Transcriptome analysis revealed 86 genes of E3s and LC-MS/MS analysis demonstrated 16 proteins of E3s. Among these, seven E3s (BRCA1, UBR4, BRE1B, UHRF1, NEDD4, CUL5, and FBX6) were shared. RT-PCR demonstrated mRNA expressions of the seven identified E3s in the kidney, and immunoblotting demonstrated changes in protein abundance of the selected E3s (BRE1B, NEDD4, and CUL5) in response to dDAVP stimulation/withdrawal or lithium-induced nephrogenic diabetes insipidus. The rate of AQP2 degradation was retarded in mpkCCDc14 cells with small interfering RNA-mediated knockdown of NEDD4 or CUL5. Taken together, identified E3s could be involved in the degradation of proteins associated with vasopressin-induced urine concentration.

Properties of lamin A mutants found in Emery-Dreifuss muscular dystrophy, cardiomyopathy and Dunnigan-type partial lipodystrophy

2001 ◽  
Vol 114 (24) ◽  
pp. 4435-4445 ◽  
Author(s):  
Cecilia Östlund ◽  
Gisèle Bonne ◽  
Ketty Schwartz ◽  
Howard J. Worman
Keyword(s):  
Muscular Dystrophy ◽  
Dilated Cardiomyopathy ◽  
Lamin A ◽  
Wild Type ◽  
Yeast Two Hybrid ◽  
Partial Lipodystrophy ◽  
Lamin B1 ◽  
The Stability ◽  
Mutant Forms ◽  
Two Hybrid

Autosomal dominant Emery-Dreifuss muscular dystrophy is caused by mutations in the LMNA gene, which encodes lamin A and lamin C. Mutations in this gene also give rise to limb girdle muscular dystrophy type 1B, dilated cardiomyopathy with atrioventricular conduction defect and Dunnigan-type partial lipodystrophy. The properties of the mutant lamins that cause muscular dystrophy, lipodystrophy and dilated cardiomyopathy are not known. We transfected C2C12 myoblasts with cDNA encoding wild-type lamin A and 15 mutant forms found in patients affected by these diseases. Immunofluorescence microscopy showed that four mutants, N195K, E358K, M371K and R386K, could have a dramatically aberrant localization, with decreased nuclear rim staining and formation of intranuclear foci. The distributions of endogenous lamin A/C, lamin B1 and lamin B2 were also altered in cells expressing these four mutants and three of them caused a loss of emerin from the nuclear envelope. In the yeast two-hybrid assay, the 15 lamin A mutants studied interacted with themselves and with wild-type lamin A and lamin B1. Pulse-chase experiments showed no decrease in the stability of several representative lamin A mutants compared with wild-type. These results indicate that some lamin A mutants causing disease can be aberrantly localized, partially disrupt the endogenous lamina and alter emerin localization, whereas others localize normally in transfected cells.

scholarly journals Ripply2 recruits proteasome complex for Tbx6 degradation to define segment border during murine somitogenesis

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Wei Zhao ◽  
Masayuki Oginuma ◽  
Rieko Ajima ◽  
Makoto Kiso ◽  
Akemi Okubo ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Es Cells ◽  
Mass Spectrometry Analysis ◽  
Expression Domain ◽  
Spectrometry Analysis ◽  
T Box ◽  
Induction System ◽  
Segmentation Boundary ◽  
The Stability

The metameric structure in vertebrates is based on the periodic formation of somites from the anterior end of the presomitic mesoderm (PSM). The segmentation boundary is defined by the Tbx6 expression domain, whose anterior limit is determined by Tbx6 protein destabilization via Ripply2. However, the molecular mechanism of this process is poorly understood. Here, we show that Ripply2 directly binds to Tbx6 in cultured cells without changing the stability of Tbx6, indicating an unknown mechanism for Tbx6 degradation in vivo. We succeeded in reproducing in vivo events using a mouse ES induction system, in which Tbx6 degradation occurred via Ripply2. Mass spectrometry analysis of the PSM-fated ES cells revealed that proteasomes are major components of the Ripply2-binding complex, suggesting that recruitment of a protein-degradation-complex is a pivotal function of Ripply2. Finally, we identified a motif in the T-box, which is required for Tbx6 degradation independent of binding with Ripply2 in vivo.

scholarly journals Circadian time series proteomics reveals daily dynamics in cartilage physiology

2019 ◽  
Author(s):  
Michal Dudek ◽  
Constanza Angelucci ◽  
Jayalath P.D. Ruckshanthi ◽  
Ping Wang ◽  
Venkatesh Mallikarjun ◽  
...  
Keyword(s):  
Time Series ◽  
Articular Cartilage ◽  
Meta Analysis ◽  
Treatment Strategies ◽  
Time Of Day ◽  
Mass Spectrometry Analysis ◽  
Protein Abundance ◽  
Spectrometry Analysis ◽  
Proteomics Study ◽  
Cartilage Biology

AbstractObjectivesArticular cartilage undergoes cyclical heavy loading and low load recovery during the 24-hour day/night cycle. We investigated the daily changes of protein abundance in mouse femoral head articular cartilage by performing 24-hour time-series proteomics study.MethodsTandem mass spectrometry analysis was used to quantify proteins extracted from mouse cartilage. Bioinformatics analysis was performed to quantify rhythmic changes in protein abundance. Primary chondrocytes were isolated and cultured for independent validation of selected rhythmic proteins.Results145 rhythmic proteins were detected. Among these were key cartilage molecules including CCN2, MATN1, PAI-1 and PLOD1 & 2. Pathway analysis revealed that proteins related to protein synthesis, cytoskeleton and glucose metabolism exhibited time-of-day dependent peaks in their abundance. Meta-analysis of published proteomics datasets from articular cartilage revealed that numerous rhythmic proteins were dysregulated in osteoarthritis and/or ageing.ConclusionsOur circadian proteomics study revealed that articular cartilage is a much more dynamic tissue than previously thought. Chondrocytes exhibit circadian rhythms not only in gene expression but also in protein abundance. Our results clearly call for the consideration of circadian timing in understanding cartilage biology, osteoarthritis pathogenesis, treatment strategies and biomarker detection.

scholarly journals The yeast mitophagy receptor Atg32 is ubiquitinated and degraded by the proteasome

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0241576
Author(s):  
Nadine Camougrand ◽  
Pierre Vigié ◽  
Cécile Gonzalez ◽  
Stéphen Manon ◽  
Ingrid Bhatia-Kiššová
Keyword(s):  
Mitochondrial Membrane ◽  
Normal Growth ◽  
Growth Conditions ◽  
Mass Spectrometry Analysis ◽  
Outer Mitochondrial Membrane ◽  
Post Translational Modifications ◽  
Spectrometry Analysis ◽  
Lysine Residues ◽  
The Stability ◽  
Respiratory Conditions

Mitophagy, the process that degrades mitochondria selectively through autophagy, is involved in the quality control of mitochondria in cells grown under respiratory conditions. In yeast, the presence of the Atg32 protein on the outer mitochondrial membrane allows for the recognition and targeting of superfluous or damaged mitochondria for degradation. Post-translational modifications such as phosphorylation are crucial for the execution of mitophagy. In our study we monitor the stability of Atg32 protein in the yeast S. cerevisiae and show that Atg32 is degraded under normal growth conditions, upon starvation or rapamycin treatment. The Atg32 turnover can be prevented by inhibition of the proteasome activity, suggesting that Atg32 is also ubiquitinated. Mass spectrometry analysis of purified Atg32 protein revealed that at least lysine residue in position 282 is ubiquitinated. Interestingly, the replacement of lysine 282 with alanine impaired Atg32 degradation only partially in the course of cell growth, suggesting that additional lysine residues on Atg32 might also be ubiquitinated. Our results provide the foundation to further elucidate the physiological significance of Atg32 turnover and the interplay between mitophagy and the proteasome.

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