Targeting and post-translational processing of human alpha1-antichymotrypsin in BY-2 tobacco cultured cells.
The post-translational processing of human alpha(1)-antichymotrypsin (AACT) in Bright Yellow-2 (BY-2) tobacco cells was assessed in relation to the cellular compartment targeted for accumulation. As determined by pulse-chase labelling experiments and immunofluorescence microscopy, AACT sent to the vacuole or the endoplasmic reticulum (ER) was found mainly in the culture medium, similar to a secreted form targeted to the apoplast. Unexpectedly, AACT expressed in the cytosol was found in the nucleus under a stable, non-glycosylated form, in contrast with secreted variants undergoing multiple post-translational modifications during their transit through the secretory pathway. All secreted forms of AACT were N-glycosylated, with the presence of complex glycans as observed naturally on human AACT. Proteolytic trimming was also observed for all secreted variants, both during their intracellular transit and after their secretion in the culture medium. Overall, the targeting of human AACT to different compartments of BY-2 tobacco cells led to the production of two protein products: (i) a stable, non-glycosylated protein accumulated in the nucleus; and (ii) a heterogeneous mixture of secreted variants resulting from post-translational N-glycosylation and proteolytic processing. Overall, these data suggest that AACT is sensitive to resident proteases in the ER, the Golgi and/or the apoplast, and that the production of intact AACT in the plant secretory pathway will require innovative approaches to protect its structural integrity in vivo. Studies are now needed to assess the activity of the different AACT variants, and to identify the molecular determinants for the nuclear localization of AACT expressed in the cytosol.
Benchabane
Meriem
M
Département de Phytologie, Pavillon des Services-INAF, Université Laval, Québec, QC, Canada, G1V 0A6.
Saint-Jore-Dupas
Claude
C
Bardor
Muriel
M
Faye
Loïc
L
Michaud
Dominique
D
Gomord
Véronique
V
D002478:Cells, Cultured; D003470:Culture Media; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000378:metabolism; D055571:Secretory Pathway; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*; D014170:Transformation, Genetic; D000514:alpha 1-Antichymotrypsin / Q000378:metabolism*
D003470:Culture Media; D011994:Recombinant Proteins; D000514:alpha 1-Antichymotrypsin