ABSTRACT
The prion protein (PrP) displays some unusual features in its biogenesis. In cell-free systems it can be synthesized as either an integral transmembrane protein spanning the membrane twice, with both amino and carboxyl domains in the lumen of the endoplasmic reticulum, or as a fully translocated polypeptide. A charged, extracytoplasmic region, termed 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 of the transmembrane form have not been identified previously. Moreover, the relationship of transmembrane and fully translocated forms of PrP 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 nascent PrP to span the membrane a second time, with its carboxyl-terminal domain in the endoplasmic reticulum lumen. Surprisingly, PrP carboxyl-terminal domain translocation does not appear to be directed by an internal signal or signal-anchor sequence located downstream of TM1, as would have been expected from studies of other multispanning membrane proteins. Rather, carboxyl-terminal domain translocation appears to be another consequence of the action of STE-TM1, that is, the same sequence responsible for generating the first membrane-spanning region. Studies of an STE-TM1-containing protein chimera in Xenopus oocytes demonstrate that most of these chains upon completion of their translation, 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 metastable "transmembrane" intermediate, residing within the aqueous environment of the translocation channel, can be converted into either the integrated transmembrane or the fully translocated form of PrP, perhaps directed by trans-acting factor (s). Such a model may explain why stable the transmembrane isoform of PrP has not been observed in normal cells and how nascent PrP might be directed to alternate pathways of folding.
