Stable and transient expression of chimeric peroxisomal membrane proteins induces an independent "zippering" of peroxisomes and an endoplasmic reticulum subdomain

Peroxisomal ascorbate peroxidase (APX) (EC 1. 11. 1. 11) was shown recently to sort through a subdomain of the ER (peroxisomal endoplasmic reticulum; pER), and in certain cases, alter the distribution and/or morphology of per oxisomes and pER when overexpressed transiently in Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) cells. Our goal was to gain insight into the dynami cs of peroxisomal membrane protein sorting by characterizing the structure and formation of reorganized peroxisomes and pER. Specifically, we test dir ectly the hypothesis that the observed phenomenon is due to the oligomeriza tion of cytosol-facing, membrane-bound polypeptides, a process referred to as membrane "zippering". Results from differential detergent permeabilizati on experiments confirmed that peroxisomal APX is a C-terminal "tail-anchore d" (C-matrix-N-cytosol) membrane protein with a majority of the polypeptide facing the cytosol. Transient expression of several APX chimeras whose pas senger polypeptides can form dimers or trimers resulted in the progressive formation of "globular" peroxisomes and circular pER membranes. Stable expr ession of the trimer-capable fusion protein yielded suspension cultures tha t reproducibly maintained a high degree of peroxisomal globules but relativ ely few detectable pER membranes. Electron micrographs revealed that the gl obules consisted of numerous individual peroxisomes, seemingly in direct co ntact with other peroxisomes and/or mitochondria. These peroxisomal cluster s or aggregates were not observed in cells transiently expressing monomeric versions of APX. These findings indicate that the progressive, independent "zippering" of peroxisomes and pER is due to the post-sorting oligomerizat ion of monomeric, cytosol-facing polypeptides that are integrally inserted into the membranes of "like" organelles. The dynamics of this process are d iscussed, especially with respect to the involvement of the microtubule cyt oskeleton.