The sorting signals for peroxisomal membrane-bound ascorbate peroxidase are within its C-terminal tail

Peroxisomal ascorbate peroxidase (APX) is a carboxyl tail-anchored, type II (N-cytosol-C-matrix) integral membrane protein that functions in the regen eration of NAD(+) in glyoxysomes of germinated oilseeds and protection of p eroxisomes in other organisms from toxic H2O2. Recently we showed that cott onseed peroxisomal APX was sorted post-translationally from the cytosol to peroxisomes via a novel reticular/circular membranous network that was inte rpreted to be a subdomain of the endoplasmic reticulum (ER), named peroxiso mal ER (pER), Here we report on the molecular signals responsible for sorti ng peroxisomal APX Deletions or site-specific substitutions of certain amin o acid residues within the hydrophilic C-terminal-most eight-amino acid res idues (includes a positively charged domain found in most peroxisomal integ ral membrane-destined proteins) abolished sorting of peroxisomal APX to per oxisomes via pER. However, the C-terminal tail was not sufficient for sorti ng chloramphenicol acetyltransferase to peroxisomes via pER, whereas the pe ptide plus most of the immediately adjacent 21-amino acid transmembrane dom ain (TMD) of peroxisomal APX was sufficient for sorting. Replacement of the peroxisomal APX TMD with an artificial TMD (devoid of putative sorting seq uences) plus the peroxisomal APX C-terminal tail also sorted chloramphenico l acetyltransferase to peroxisomes via pER, indicating that the peroxisomal APX TMD does not possess essential sorting information. Instead, the TMD a ppears to confer the proper context required for the conserved positively c harged domain to function within peroxisomal APX as an overlapping pER sort ing signal and a membrane peroxisome targeting signal type 2.