DETERMINANTS OF CARBOXYL-TERMINAL DOMAIN TRANSLOCATION DURING PRION PROTEIN BIOGENESIS

The prion protein (PrP) displays some unusual features in its biogenes is. In cell-free systems it can be synthesized as either an integral t ransmembrane protein spanning the membrane twice, with both amino and carboxyl domains in the lumen of the endoplasmic reticulum, or as a fu lly translocated polypeptide, A charged, extracytoplasmic region, term ed the Stop Transfer Effector (STE) sequence, has been shown to direct the nascent translocating chain to stop at the adjoining hydrophobic domain to generate the first membrane-spanning region (TM1). However, the determinants of the second translocation event in the biogenesis o f the transmembrane form have not been identified previously. Moreover , the relationship of transmembrane and fully translocated forms of Pr P has not been well understood. Here, we report progress in resolving both of these issues. Using protein chimeras in cell-free translation systems and Xenopus oocytes, we identify the sequence which directs na scent PrP to span the membrane a second time, with its carboxyl-termin al domain in the endoplasmic reticulum lumen. Surprisingly, PrP carbox yl-terminal domain translocation does not appear to be directed by an internal signal or signal-anchor sequence located downstream of TM1, a s would have been expected from studies of other multispanning membran e proteins. Rather, carboxyl-terminal domain translocation appears to be another consequence of the action of STE-TM1, that is, the same seq uence responsible for generating the first membrane-spanning region. S tudies of an STE-TM1-containing protein chimera in Xenopus oocytes dem onstrate that most of these chains upon completion of their translatio n, initially span the membrane twice, with a topology similar to that of transmembrane PrP, but are carbonate-extractable. These chains have the transmembrane orientation only transiently and chase into a fully translocated form. These results support a model in which a metastabl e ''transmembrane'' intermediate, residing within the aqueous environm ent of the translocation channel, can be converted into either the int egrated transmembrane or the fully translocated form of PrP, perhaps d irected by trans-acting factor (s). Such a model may explain why stabl e the transmembrane isoform of PrP has not been observed in normal cel ls and how nascent PrP might be directed to alternate pathways of fold ing.