Translocation efficiency of apolipoprotein B is determined by the presence of beta-sheet domains, not pause transfer sequences.
Cotranslational translocation of apoB100 across the endoplasmic reticulum (ER) membrane is inefficient, resulting in exposure of nascent apoB on the cytosolic surface of the ER. This predisposes apoB100 to ubiquitinylation and targeting for proteasomal degradation. It has been suggested that pause transfer sequences (PTS) present throughout apoB cause inefficient translocation. On the other hand, our previous study demonstrated that the translocation efficiency of apoB100 is dependent on the presence of a beta-sheet domain between 29 and 34% of full-length apoB100 (Liang, J.-S., Wu, X., Jiang, H., Zhou, M., Yang, H., Angkeow, P., Huang, L.-S., Sturley, S. L., and Ginsberg, H. N. (1998) J. Biol. Chem. 273, 35216-35221); this region of apoB has no PTS. However, the effects of the beta-sheet domain may require the presence of PTS elsewhere in the N-terminal region of apoB100. To further investigate the roles of PTS and beta-sheet domains in the translocation of apoB100 across the ER, we transfected McArdle RH7777, HepG2, or Chinese hamster ovary cells with human albumin (ALB)/human apoB chimeric cDNA constructs: ALB/B12-17 (two PTS but no beta-sheet), ALB/B29-34 (beta-sheet but no PTS), ALB/B36-41 (two PTS and a beta-sheet), and ALB/B49-54 (neither PTS nor a beta-sheet). ALB/ALB1-40 served as a control. Compared with ALB/ALB1-40, secretion rates of ALB/B12-17, ALB/B29-34, and ALB/B36-41 were reduced. Secretion of ALB/B49-54 was similar to that of ALB/ALB1-40. However, only ALB/B29-34 and ALB/B36-41 had increased proteinase K sensitivity, ubiquitinylation, and increased physical interaction with Sec61alpha. These results indicate that the translocation efficiency of apoB100 is determined mainly by the presence of beta-sheet domains. PTS do not appear to affect translocation, but may affect secretion by other mechanisms.
Yamaguchi
Junji
J
Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA.
Conlon
Donna M
DM
Liang
John J
JJ
Fisher
Edward A
EA
Ginsberg
Henry N
HN
D000818:Animals; D053299:Apolipoprotein B-100; D001055:Apolipoproteins B / Q000737:chemistry / Q000378:metabolism*; D016466:CHO Cells; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D019286:Endopeptidase K / Q000737:chemistry; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006801:Humans; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D025801:Ubiquitin / Q000737:chemistry
D053299:Apolipoprotein B-100; D001055:Apolipoproteins B; D018076:DNA, Complementary; D025801:Ubiquitin; D019286:Endopeptidase K