Compartmentalization of the plant peroxin, AtPex10p, within subdomain(s) of ER

A family of proteins collectively referred to as ‘‘peroxins’’ are involved variously in the biogenesis of peroxisomes. Recent studies on the characterization of a T-DNA mutant of a putative plant peroxin gene in Arabidopsis, AtPEX10, led to conclusions that the peroxin gene product, AtPex10p, is required for normal embryo development and viability, possibly through its involvement in the formation of protein bodies, oleosomes, and peroxisomes from segments of ER. Because the homozygous condition was lethal during seed embryogenesis, Arabidopsis suspension cells were used in the current study for determining the subcellular localization of AtPex10p. Surprisingly, endogenous AtPex10p was not found via immunofluorescence microscopy in peroxisomes; instead, it was observed throughout non-vacuolar cytoplasm partially colocalized with ER marker proteins. Transiently overexpressed AtPex10p (epitope-tagged and fluorescent protein-fused) did not sort to peroxisomes or ER; all constructs accumulated in non-organellar cytosol. Electron immunogold microscopy of the suspension cells confirmed the ER, but not peroxisomal, localization of AtPex10p. Additional evidence for ER localization was obtained from cell fractionation studies. AtPex10p was found exclusively in calnexin-enriched (ER), not catalase-enriched (peroxisomal), regions of sucrosedensity gradients. Also, microsomal calnexin and AtPex10p exhibited concomitant magnesium shifts in sucrose gradients, characteristic of localization in rough ER-derived vesicles. AtPex10p in these vesicles was KCl insoluble, alkaline-carbonate soluble, and protease-digestible in the absence of detergent. Collective results indicate that AtPex10p is a cytosolic-facing, peripherally associated membrane protein that occurs in relatively low abundance within portion(s) (subdomains) of ER and possibly in vesicles where it participates in peroxisome formation as an ‘‘early peroxin’’. # 2004 Elsevier Ireland Ltd. All rights reserved.