Molecular characterization of two plant BI-1 homologues which suppress Bax-induced apoptosis in human 293 cells.
To date, few homologues of animal programmed cell death (PCD) regulators have been identified in plants. Among these is the plant Bax Inhibitor-1 (BI-1) protein, which possesses, like its human counterpart, the ability to suppress Bax-induced lethality in yeast cells. As the role of BI-1 in the regulation of plant PCD remains to be elucidated, we cloned BnBI-1 and NtBI-1 from cDNA libraries of oilseed rape ( Brassica napus L.) and tobacco ( Nicotiana tabacum L.). The analysis of the deduced amino acid sequences of BnBI-1 and NtBI-1 indicated that these proteins share a relatively high level of identity with other plant BI-1 proteins (73-95%) as well as with animal BI-1 proteins (26-42%). Comparative analysis with other available plant BI-1 proteins allowed the establishment of a structural model presenting seven transmembrane domains. Moreover, transient co-transfection of Bax with BnBI-1 or NtBI-1 in human embryonic kidney 293 cells revealed that both proteins can substantially inhibit apoptosis induced by Bax overexpression. Localization studies were also conducted using stable transformation of tobacco BY-2 cells and Saccharomyces cerevisiae, or transient expression in tobacco leaves, with the fusion protein BnBI-1GFP under control of the cauliflower mosaic virus 35S promoter. All transformants showed a fluorescence pattern of distribution typical of an endoplasmic reticulum (ER) protein. Results from differential permeabilization experiments in BY-2 cells expressing BnBI-1GFP also showed that the C-terminus is located on the cytosolic side of the ER. Taken altogether, our results suggest that BI-1 is evolutionarily conserved and could act as a key regulator of a death pathway common to plants and animals.
Bolduc
Nathalie
N
Department of Biochemistry and Microbiology, Life and Health Sciences Research Building, Laval University, Québec, G1K 7P4, Canada.
Ouellet
Mario
M
Pitre
Frédéric
F
Brisson
Louise F
LF
D000595:Amino Acid Sequence; D017209:Apoptosis / Q000235:genetics* / Q000502:physiology; D051017:Apoptosis Regulatory Proteins; D029688:Brassica napus / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D007668:Kidney / Q000166:cytology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D011506:Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism
D051017:Apoptosis Regulatory Proteins; D008164:Luminescent Proteins; D008565:Membrane Proteins; D011506:Proteins; C484209:TMBIM6 protein, human; D049452:Green Fluorescent Proteins