Limits...
Characterization of the Arabidopsis thaliana E3 ubiquitin-ligase AtSINAL7 and identification of the ubiquitination sites.

Peralta DA, Araya A, Nardi CF, Busi MV, Gomez-Casati DF - PLoS ONE (2013)

Bottom Line: We also found that higher AtSINAL7 transcript levels are present in tissues undergoing active cell division during floral development.An interesting observation is the circadian expression pattern of AtSINAL7 mRNA in floral buds.Furthermore, UV-B irradiation induces the expression of this transcript indicating that AtSINAL7 may be involved in a wide range of different cell processes.

View Article: PubMed Central - PubMed

Affiliation: Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI-CONICET), Universidad Nacional de Rosario, Rosario, Argentina.

ABSTRACT
Protein ubiquitination leading to degradation by the proteasome is an important mechanism in regulating key cellular functions. Protein ubiquitination is carried out by a three step process involving ubiquitin (Ub) activation by a E1 enzyme, the transfer of Ub to a protein E2, finally an ubiquitin ligase E3 catalyzes the transfer of the Ub peptide to an acceptor protein. The E3 component is responsible for the specific recognition of the target, making the unveiling of E3 components essential to understand the mechanisms regulating fundamental cell processes through the protein degradation pathways. The Arabidopsis thaliana seven in absentia-like 7 (AtSINAL7) gene encodes for a protein with characteristics from a C3HC4-type E3 ubiquitin ligase. We demonstrate here that AtSINAL7 protein is indeed an E3 protein ligase based on the self-ubiquitination in vitro assay. This activity is dependent of the presence of a Lys residue in position 124. We also found that higher AtSINAL7 transcript levels are present in tissues undergoing active cell division during floral development. An interesting observation is the circadian expression pattern of AtSINAL7 mRNA in floral buds. Furthermore, UV-B irradiation induces the expression of this transcript indicating that AtSINAL7 may be involved in a wide range of different cell processes.

Show MeSH
Expression analysis of recombinant AtSINAL7.The E. coli cell extracts electrophoresed on SDS-PAGE were revealed with Coomassie brilliant blue staining. Lane 1: Protein extract from uninduced bacteria. Lane 2: Soluble proteins obtained after 5 h IPTG induced bacterial culture. Lane 3: Purified AtSINAL7 fraction stained with Coomassie blue. Lane 4: Western blott analysis of the purified AtSINAL7 fraction revealed using anti-His antobodies. MW (kDa), PageRuler Prestained Protein Ladder (Fermentas).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3756039&req=5

pone-0073104-g001: Expression analysis of recombinant AtSINAL7.The E. coli cell extracts electrophoresed on SDS-PAGE were revealed with Coomassie brilliant blue staining. Lane 1: Protein extract from uninduced bacteria. Lane 2: Soluble proteins obtained after 5 h IPTG induced bacterial culture. Lane 3: Purified AtSINAL7 fraction stained with Coomassie blue. Lane 4: Western blott analysis of the purified AtSINAL7 fraction revealed using anti-His antobodies. MW (kDa), PageRuler Prestained Protein Ladder (Fermentas).

Mentions: To characterize the seven in absentia like 7 protein from Arabidopsis thaliana, the DNA fragment containing the AtSINAL7 coding sequence (286 codons) was fused to an N-terminal His6-tag when cloned onto pRSETb expression vector. The recombinant protein was purified using a HisTrap chelating affinity chromatography after expression in E. coli (BL21) pLys strain. The purified recombinant AtSINAL7 protein of 32 KDa was successfully induced (Figure 1, lane 2) and purified to homogeneity as shown by protein staining (lane 3). The presence of recombinant AtSINAL7 was assessed by Western blot analysis using with the anti-His antibody (lane 4).


Characterization of the Arabidopsis thaliana E3 ubiquitin-ligase AtSINAL7 and identification of the ubiquitination sites.

Peralta DA, Araya A, Nardi CF, Busi MV, Gomez-Casati DF - PLoS ONE (2013)

Expression analysis of recombinant AtSINAL7.The E. coli cell extracts electrophoresed on SDS-PAGE were revealed with Coomassie brilliant blue staining. Lane 1: Protein extract from uninduced bacteria. Lane 2: Soluble proteins obtained after 5 h IPTG induced bacterial culture. Lane 3: Purified AtSINAL7 fraction stained with Coomassie blue. Lane 4: Western blott analysis of the purified AtSINAL7 fraction revealed using anti-His antobodies. MW (kDa), PageRuler Prestained Protein Ladder (Fermentas).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3756039&req=5

pone-0073104-g001: Expression analysis of recombinant AtSINAL7.The E. coli cell extracts electrophoresed on SDS-PAGE were revealed with Coomassie brilliant blue staining. Lane 1: Protein extract from uninduced bacteria. Lane 2: Soluble proteins obtained after 5 h IPTG induced bacterial culture. Lane 3: Purified AtSINAL7 fraction stained with Coomassie blue. Lane 4: Western blott analysis of the purified AtSINAL7 fraction revealed using anti-His antobodies. MW (kDa), PageRuler Prestained Protein Ladder (Fermentas).
Mentions: To characterize the seven in absentia like 7 protein from Arabidopsis thaliana, the DNA fragment containing the AtSINAL7 coding sequence (286 codons) was fused to an N-terminal His6-tag when cloned onto pRSETb expression vector. The recombinant protein was purified using a HisTrap chelating affinity chromatography after expression in E. coli (BL21) pLys strain. The purified recombinant AtSINAL7 protein of 32 KDa was successfully induced (Figure 1, lane 2) and purified to homogeneity as shown by protein staining (lane 3). The presence of recombinant AtSINAL7 was assessed by Western blot analysis using with the anti-His antibody (lane 4).

Bottom Line: We also found that higher AtSINAL7 transcript levels are present in tissues undergoing active cell division during floral development.An interesting observation is the circadian expression pattern of AtSINAL7 mRNA in floral buds.Furthermore, UV-B irradiation induces the expression of this transcript indicating that AtSINAL7 may be involved in a wide range of different cell processes.

View Article: PubMed Central - PubMed

Affiliation: Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI-CONICET), Universidad Nacional de Rosario, Rosario, Argentina.

ABSTRACT
Protein ubiquitination leading to degradation by the proteasome is an important mechanism in regulating key cellular functions. Protein ubiquitination is carried out by a three step process involving ubiquitin (Ub) activation by a E1 enzyme, the transfer of Ub to a protein E2, finally an ubiquitin ligase E3 catalyzes the transfer of the Ub peptide to an acceptor protein. The E3 component is responsible for the specific recognition of the target, making the unveiling of E3 components essential to understand the mechanisms regulating fundamental cell processes through the protein degradation pathways. The Arabidopsis thaliana seven in absentia-like 7 (AtSINAL7) gene encodes for a protein with characteristics from a C3HC4-type E3 ubiquitin ligase. We demonstrate here that AtSINAL7 protein is indeed an E3 protein ligase based on the self-ubiquitination in vitro assay. This activity is dependent of the presence of a Lys residue in position 124. We also found that higher AtSINAL7 transcript levels are present in tissues undergoing active cell division during floral development. An interesting observation is the circadian expression pattern of AtSINAL7 mRNA in floral buds. Furthermore, UV-B irradiation induces the expression of this transcript indicating that AtSINAL7 may be involved in a wide range of different cell processes.

Show MeSH