Using Deubiquitylating Enzymes as Research Tools

Self Cite

121

132

Background: Glutathionylation is a major post-translational modification that regulates protein function. Results: Human glutathione transferase Omega 1 (GSTO1-1) can catalyze the deglutathionylation of protein thiols in vitro and in cell culture. Conclusion: GSTO1-1, but not GSTO2-2, catalyzes the deglutathionylation of actin and other proteins under physiological conditions. Significance: Genetic variation in GSTO1-1-catalyzed deglutathionylation may influence the function of multiple proteins in signaling pathways.

Section: Methodsmentioning

confidence: 99%

A Role for Glutathione Transferase Omega 1 (GSTO1-1) in the Glutathionylation Cycle

Menon

Board

2013

Self Cite

121

132

“…Two point mutations, Y176G and A177Y, were inserted into AtCpn20 using the QuikChange protocol (Stratagene) to give AtCpn20 mut . All cofactor genes were cloned between the SacII-HindIII sites of the vector pHue and expressed as cleavable His 6 -ubiquitin fusions that resulted in native N termini (25,26).…”

Section: Methodsmentioning

confidence: 99%

“…The resin was washed with buffer A, 25 mM imidazole before eluting the His 6 -ubiquitin fusion protein with buffer A, 200 mM imidazole. The ubiquitin moiety was cleaved overnight at 20°C using the deubiquitylating enzyme Usp2 (26). The protein was dialyzed against buffer B (20 mM Tris-HCl, pH 7.5, 20 mM NaCl, 1 mM EDTA), applied to a pre-equilibrated Mono Q 16/10 HR column (GE Healthcare), and eluted using a linear salt gradient to 0.5 M NaCl.…”

Section: Methodsmentioning

confidence: 99%

Chaperonin Cofactors, Cpn10 and Cpn20, of Green Algae and Plants Function as Hetero-oligomeric Ring Complexes

Tsai

Mueller‐Cajar

Saschenbrecker

et al. 2012

Background:The chloroplast chaperonin system is encoded by multiple genes. Results: The chaperonin cofactors of the green alga C. reinhardtii and the plant A. thaliana form hetero-oligomeric ring complexes containing seven ϳ10-kDa modules. Conclusion:The hetero-oligomeric cofactors were able to interact with chaperonin and assist protein folding. Significance: Formation of hetero-oligomers can explain the occurrence of multiple chaperonin cofactor genes in chloroplasts.

“…These cells were co-transformed with pACYCUb-M35, which directs expression of an N-terminally tagged His 6 -ubiquitin-M35 fusion (H6-Ub-M35). This was made by amplifying the M35 coding region with primers 5Ј-SacIIccmM1 (5Ј-ctccgcggtggaatgagcgcttataacggccaaggccgactc) and 3Ј-HindIIIccmM (5Ј-aagcttacggcttttgaatcaacagttc) and cloning the SacII-HindIII fragment into plasmid pHue (25) and then ligating the 1263-bp NcoI-HindIII H6-Ub-M35 coding sequence downstream of the trc promoter in pACYCtrc (24).…”

Section: Expression Of N-and C-terminally Tagged His 6 Ccmmmentioning

confidence: 99%

“…Protein and chlorophyll content of extracts was determined by the methods of Bradford (28) and Porra et al (31), respectively. Synechococcus PCC7942 Rubisco small subunits (RbcS) used in complementation studies were purified using the procedure described previously (25).…”

mentioning

confidence: 99%

Analysis of Carboxysomes from Synechococcus PCC7942 Reveals Multiple Rubisco Complexes with Carboxysomal Proteins CcmM and CcaA

Long

Badger

Whitney

et al. 2007

173

224

In cyanobacteria, the key enzyme for photosynthetic CO 2 fixation, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), is bound within proteinaceous polyhedral microcompartments called carboxysomes. Cyanobacteria with Form IB Rubisco produce ␤-carboxysomes whose putative shell proteins are encoded by the ccm-type genes. To date, very little is known of the protein-protein interactions that form the basis of ␤-carboxysome structure. In an effort to identify such interactions within the carboxysomes of the ␤-cyanobacterium Synechococcus sp. PCC7942, we have used polyhistidine-tagging approaches to identify at least three carboxysomal subcomplexes that contain active Rubisco. In addition to the expected L 8 S 8 Rubisco, which is the major component of carboxysomes, we have identified two Rubisco complexes containing the putative shell protein CcmM, one of which also contains the carboxysomal carbonic anhydrase, CcaA. The complex containing CcaA consists of Rubisco and the full-length 58-kDa form of CcmM (M58), whereas the other is made up of Rubisco and a short 35-kDa form of CcmM (M35), which is probably translated independently of M58 via an internal ribosomal entry site within the ccmM gene. We also show that the high CO 2 -requiring ccmM deletion mutant (⌬ccmM) can achieve nearly normal growth rates at ambient CO 2 after complementation with both wild type and chimeric (His 6 -tagged) forms of CcmM. Although a significant amount of independent L 8 S 8 Rubisco is confined to the center of the carboxysome, we speculate that the CcmMCcaA-Rubisco complex forms an important assembly coordination within the carboxysome shell. A speculative carboxysome structural model is presented.Prokaryotic organisms are distinct from their eukaryotic counterparts by the absence of specialized membrane-bound compartments. Although it is commonly considered that prokaryotic cells do not compartmentalize cellular processes, there is growing evidence that cyanobacteria and some ␤-proteobacteria have the means to do so when certain enzymes need to be sequestered or protected for maximal catalytic activity (1-3). Carboxysomes, for example, are polyhedral protein microcompartments found in cyanobacteria and chemoautotrophic bacteria, which contain the majority of a cell's ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) (4). These microcompartments are essential to the operation of the CO 2 -concentrating mechanism in cyanobacteria. The CO 2 -concentrating mechanism utilizes a number of inorganic carbon (C i , the sum of CO 2 and HCO 3 Ϫ ) transport systems to elevate cellular HCO 3 Ϫ concentrations prior to CO 2 fixation (for reviews, see Refs. 5 and 6). Rubisco is a hexadecameric enzyme with a core of eight large subunits (L 8 ) made up of four L 2 dimers, each containing two catalytic sites. Attached to each end of the L 8 core are four small subunits (S 8 ) that are necessary for catalytic competence (7). Rubisco carries out the CO 2 fixation process in the initial step of the Calvin cycle, yet it is characterized by ...