STUDIES ON THE TRANSLOCATION OF THE AMINO-TERMINUS OF APOLIPOPROTEIN-B INTO THE ENDOPLASMIC-RETICULUM

Apolipoprotein (apo) B is either co-translationally assembled into lip oproteins, or becomes associated with the membrane of the endoplasmic reticulum (ER) and is subsequently degraded, It has been proposed that apoB undergoes a novel process of translocation which generates cytop lasmically exposed apoB in the ER of hepatic and non-hepatic cells. Tr ansmembrane forms of apoB can also be generated by in vitro translatio n (Chuck, S. L., and Lingappa, V. R. (1992) Cell 68, 9-21), which migh t explain the origin of untranslocated apoB in vivo. Here we have inve stigated a protocol which generates transmembrane forms of apoB during in vitro translation of truncated RNA transcripts, We observe that ap oB can become transmembrane at sites of ribosome pausing and be held i n this configuration by persistence of tRNA on the peptide chains. Rib osome pausing also occurs at these same sites in the absence of accept or microsomes. Transmembrane topology can be generated at sites of rib osome pausing in a cytosolic protein, sea urchin cyclin when fused to a signal sequence, Mapping of the ribosome pause sites in apoB and in cyclin revealed no amino acid sequence homology, Chimeric constructs w ith engineered downstream glycosylation sites showed no evidence that ribosome pause sequences affect translocation of transcripts with term ination codons. Transmembrane forms of apoB and cyclin were not genera ted during translocation into the ER in transfected COS cells.