Systematic expression of the complete coding sequence of apoB-100 does not reveal transmembrane determinants.
We have investigated the hypothesis that apolipoprotein B undergoes a regulated process of translocation into the endoplasmic reticulum (ER) which causes the protein to adopt a transmembrane configuration. Protein segments representing the complete coding sequence of apolipoprotein B were first expressed by in vitro translation of transcripts from seven overlapping transcripts. Two regions were identified (located at residue 2425 and between residues 4149 and 4348) that can undergo incomplete translocation into pancreatic microsomes. Ribosome pausing at these sites uncoupled translation from translocation, leading to the synthesis of large cytoplasmically oriented segments of protein. In contrast, when these two regions were expressed by transfection in cultured cells, transmembrane structures were not detected. Endogenous apolipoprotein B-100 synthesis in HepG2 cells generates a spectrum of nascent chains, indicating that ribosome pausing can also occur in intact cells. However, the cellular pause products were cotranslationally translocated. While endogenous apolipoprotein B-100 in HepG2 cells was fully translocated, discrete proteolytic fragments were generated from the amino terminus of the protein when proteases gained access to the lumen of permeabilized microsomes. These products were similar in size and sequence to apoliprotein B proteolytic fragments previously ascribed as the luminal domains of transmembrane apoB-100 molecules (Du, E. Z., Kurth, J., Wang, S. L., Humiston, P., and Davis, R. A. 1994. J. Biol. Chem. 269: 24169-24176).
Medical Research Council Molecular Medicine Group, Royal Postgraduate Medical School, London, United Kingdom.
J M
JM
Leiper
G B
GB
Harrison
J
J
Bayliss
J D
JD
Scott
R J
RJ
Pease
D000595:Amino Acid Sequence; D000818:Animals; D016632:Apolipoprotein A-I / Q000737:chemistry; D053299:Apolipoprotein B-100; D001055:Apolipoproteins B / Q000737:chemistry* / Q000235:genetics*; D001483:Base Sequence; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism*; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D007163:Immunosorbent Techniques; D008862:Microsomes, Liver / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D014176:Protein Biosynthesis; D012503:Saponins / Q000494:pharmacology; D014162:Transfection
D016632:Apolipoprotein A-I; D053299:Apolipoprotein B-100; D001055:Apolipoproteins B; D010446:Peptide Fragments; D012503:Saponins