REASSESSMENT OF THE SUBCELLULAR-LOCALIZATION OF P63

p63 is a type II integral membrane protein that has previously been su ggested to be a resident protein of a membrane network interposed betw een the ER and the Golgi apparatus. In the present study, we have prod uced a polyclonal antibody against the purified human p63 protein to r eassess the subcellular distribution of p63 by confocal immunofluoresc ence, immunoelectron microscopy, and cell fractionation, Double immuno fluorescence of COS cells showed significant colocalization of p63 and a KDEL-containing lumenal ER marker protein, except for differences i n the staining of the outer nuclear membrane. Immunoelectron microscop y of native HepG2 cells and of COS cells transfected with p63 revealed that both endogenous and overexpressed p63 are predominantly localize d in the rough ER. While p63 was colocalized with protein disulfide is omerase, an ER marker protein, very little overlap of p63 was found wi th ERGIC-53, an established marker for the ER-Golgi intermediate compa rtment. When rough and smooth membranes were prepared from rat liver, p63 was found to copurify with ribophorin II, a rough ER protein, Both p63 and ribophorin II were predominantly recovered in rough microsome s and were largely separated from the intermediate compartment marker protein p58. From these results it is concluded that p63 is localized in the rough ER.