Overexpression of BPM1 deletion mutants in Arabidopsis thaliana

Proteini BPM sudjeluju u ciljanoj degradaciji proteina putem proteasoma 26S. Tijekom rasta, razvoja i u stresu, u stanici neprekidno dolazi do ciljane degradacije proteina. Pri tome ključnu ulogu ima degradacija transkripcijskih faktora koji su važni regulatori rasta i razvoja. Proteini BPM su adaptori E3 ligazi CUL3 (kulin 3) te svojim vezanjem na transkripcijske faktore sudjeluju u njihovoj ubikvitinaciji i regulaciji degradacije. Dosadašnja istraživanja su pokazala da protein BPM1 iz uročnjaka (Arabidopsis thaliana) lokalizira u jezgri i u citoplazmi transformiranih stanica duhana BY2 te je otkriven C-terminalni bipartitni nuklearni lokalizacijski signal (NLS) proteina BPM1, a u ovome je radu istražena njegova uloga u uročnjaku. U tu svrhu regenerirane su transgenične biljke uročnjaka s pojačanom ekspresijom krnjeg BPM1 proteina (kojem je deletirana C-terminalna NLS regija) fuzioniranog sa zelenim fluorescentnim proteinom (GFP), te biljke s pojačanom ekspresijom nuklearnog lokalizacijskog signala proteina BPM1 fuzioniranog s GFP-om. U transgeničnim biljkama provjerena je prisutnost transgena i njihova ekspresija, a imunodetekcijom je provjerena prisutnost rekombinantnih proteina. Fluorescentnom mikroskopijom je utvrđeno da nuklearni lokalizacijski signal proteina BPM1 fuzioniran s GFP-om (NLSbpm1-GFP) lokalizira u jezgri te da je time zaslužan za smještaj proteina BPM1 u jezgru, što je preduvjet za sudjelovanje proteina BPM1 u različitim staničnim procesima koji se u jezgri odvijaju. Sub-stanična lokalizacija krnjeg proteina BPM1 fuzioniranog s proteinom GFP (delta-3-BPM1-GFP) nije utvrđena.BPM proteins participate in targeted protein degradation via the 26S proteasome. During growth, development and stress in the cell, targeted degradation of the protein occurs continuously. The degradation of transcription factors, which are important regulators of growth and development, plays a key role in this. BPM proteins are adapters of the E3 ligase CUL3 (cullin 3) and by their binding to transcription factors participate in their ubiquitination and regulation of degradation. Studies to date have shown that the Arabidopsis thaliana BPM1 protein localizes to the nucleus and cytoplasm of transformed BY2 tobacco cells, and the C-terminal bipartite nuclear localization signal (NLS) of BPM1 protein was detected. Its role in the Arabidopsis was investigated in this paper. For this purpose, transgenic plants with enhanced expression of truncated BPM1 protein (to which the C-terminal NLS region was deleted) fused with green fluorescent protein (GFP) and plants with enhanced expression of the nuclear localization signal of BPM1 protein fused with GFP were regenerated. In transgenic plants, the presence of transgenes and their expression was checked, and the presence of recombinant proteins was checked by immunodetection. Fluorescence microscopy has determined that the nuclear localization signal of BPM1 fused with GFP (NLSbpm1-GFP) localizes to the nucleus and is thus responsible for the placement of BPM1 in the nucleus, which is a prerequisite for the involvement of BPM1 in various cellular processes in the nucleus place. Sub-cellular localization of truncated BPM1 protein fused with GFP protein (delta-3-BPM1-GFP) has not been established