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 peroxisomes and pER when overexpressed transiently in Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) cells. Our goal was to gain insight into the dynamics of peroxisomal membrane protein sorting by characterizing the structure and formation of reorganized peroxisomes and pER. Specifically, we test directly the hypothesis that the observed phenomenon is due to the oligomerization of cytosol-facing, membrane-bound polypeptides, a process referred to as membrane zippering. Results from differential detergent permeabilization experiments confirmed that peroxisomal APX is a C-terminal tail-anchored (C-matrix-N-cytosol) membrane protein with a majority of the polypeptide facing the cytosol. Transient expression of several APX chimeras whose passenger polypeptides can form dimers or trimers resulted in the progressive formation of globular peroxisomes and circular pER membranes. Stable expression of the trimer-capable fusion protein yielded suspension cultures that reproducibly maintained a high degree of peroxisomal globules but relatively few detectable pER membranes. Electron micrographs revealed that the globules consisted of numerous individual peroxisomes, seemingly in direct contact with other peroxisomes and/or mitochondria. These peroxisomal clusters 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 oligomerization of monomeric, cytosol-facing polypeptides that are integrally inserted into the membranes of like organelles. The dynamics of this process are discussed, especially with respect to the involvement of the microtubule cytoskeleton.