RESUMO
The unfolded protein response (UPR) is a homeostatic cellular response conserved in eukaryotic cells to alleviate the accumulation of unfolded proteins in the endoplasmic reticulum (ER). Arabidopsis bZIP28 is a membrane-bound transcription factor activated by proteolytic cleavage in response to ER stress, thereby releasing its cytosolic portion containing the bZIP domain from the membrane to translocate into the nucleus where it induces the transcription of genes encoding ER-resident molecular chaperones and folding enzymes. It has been widely recognized that the proteolytic activation of bZIP28 is mediated by the sequential cleavage of site-1 protease (S1P) and site-2 protease (S2P). In the present study we provide evidence that bZIP28 protein is cleaved by S2P, but not by S1P. We demonstrated that wild-type and s1p mutant plants produce the active, nuclear form of bZIP28 in response to the ER stress inducer tunicamycin. In contrast, tunicamycin-treated s2p mutants do not accumulate the active, nuclear form of bZIP28. Consistent with these observations, s2p mutants, but not s1p mutants, exhibited a defective transcriptional response of ER stress-responsive genes and significantly higher sensitivity to tunicamycin. Interestingly, s2p mutants accumulate two membrane-bound bZIP28 fragments with a shorter ER lumen-facing C-terminal domain. Importantly, the predicted cleavage sites are located far from the canonical S1P recognition motif previously described. We propose that ER stress-induced proteolytic activation of bZIP28 is mediated by the sequential actions of as-yet-unidentified protease(s) and S2P, and does not require S1P.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Pró-Proteína Convertases/metabolismo , Serina Endopeptidases/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica de Plantas , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Mutação/genética , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Pró-Proteína Convertases/genética , Serina Endopeptidases/genética , Resposta a Proteínas não Dobradas/genética , Resposta a Proteínas não Dobradas/fisiologiaRESUMO
The N-glycosylation inhibitor tunicamycin triggers endoplasmic reticulum stress response and inhibits efficient protein secretion in eukaryotes. Using Arabidopsis suspension cells, we showed that the reduced secretion of mannose-binding lectin 1 (MBL1) protein by tunicamycin is accompanied by a significant decrease in MBL1 mRNA, suggesting that mRNA destabilization is the major cause of the inhibition of protein secretion in plants.
Assuntos
Antibacterianos/farmacologia , Arabidopsis/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Lectina de Ligação a Manose/antagonistas & inibidores , Células Vegetais/efeitos dos fármacos , RNA Mensageiro/metabolismo , Tunicamicina/farmacologia , Arabidopsis/genética , Arabidopsis/metabolismo , Técnicas de Cultura de Células , Meios de Cultura , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/genética , Expressão Gênica , Glicosilação , Lectina de Ligação a Manose/genética , Lectina de Ligação a Manose/metabolismo , Células Vegetais/metabolismo , Estabilidade de RNARESUMO
Under the unfolded protein response (UPR), transcripts encoding the endoplasmic reticulum (ER) chaperones are increased and those encoding proteins synthesized in the ER are decreased. To reproducibly detect such changes of an expression profile, homogeneous growth of plants is desired. In addition, uniform treatment with drugs inducing the UPR is also necessary. Here we describe our methods of plant culture and drug treatment, and procedure to detect gene expression by quantitative RT-PCR.