Asymmetric distribution of pause transfer sequences in apolipoprotein B-I00

Lipoprotein assembly requires a complex and regulated set of events that includes apolipoprotein B (apoB) translocation across the endoplasmic reticulum (ER) membrane, folding, and association with lipids. Unlike simple secretory proteins which are cotranslationally translocated directly into the ER lumen, nascent apoB contains pause transfer (PT) sequences that direct the transient stopping and subsequent restarting of its translocation, a phenomenon termed translocational pausing. During one particular translocational pause in apoB, the ribosome-membrane junction and ER translocation channel have been shown to be altered in such a way as to expose the nascent polypeptide to the cytosol and direct a change in the proteins neighboring the nascent chain. In this study, we have experimentally identified the location and distribution of the translocational pauses that are present throughout apoB-100.P We find that pause transfer sequences are distributed asymmetrically, clustering in three distinct domains: a) nine functional PT sequences appear in the amino terminal 20% of apoB, b) four more PT sequences occur just before the end of apoB48, and c) an additional ten PT sequences are found between apoB-65-95. These clusters are interrupted by two lipid binding regions of approximately 100 kD each in which no PT sequences occur. The implications of this asymmetric distribution of PT sequences, and their correlation with previously hypothesized structural and functional domains of apoB, are discussed.Kivlen, M. H., C. A. Dorsey, V. R. Lingappa, and R. S. Hegde. Asymmetric distribution of pause transfer sequences in apolipoprotein B-100.J. Lipid Res. 1997. 38: 1149-1162. Supplementary key words esis translocation rndoplasmic reticulum topogenic sequence lipoprotein biogenApolipoprotein B (apoB) is a large (-500 kD) hydrophobic protein found in chylomicrons, very low density lipoproteins (VLDL), and low density lipoprotein (LDL) particles. Lipoprotein particle assembly is a complex and regulated set of events during which apoB undergoes both covalent modifications (e.g., glycosylation and disulfide bond formation) and noncovalent modifications (e.g., chain folding and association with lipids) ( 1, 2). At least some of these events are likely to occur during translocation of apoB across the membrane of the endoplasmic reticulum (ER), when the apoB chain is unfolded and amenable to associations or modifications that would otherwise be sterically constrained later in biogenesis. Indeed, a variety of enzymes that catalyze modifications of proteins act at the time of translocation (3-5). To explore the possibility that specialized mechanisms were involved in lipoprotein particle assembly, we previously examined the early biogenesis of apoB (6,7). We found that apoB contains specific sequences, termed pause transfer (PT) sequences, that direct the transient stopping and subsequent restarting of chain translocation into the ER lumen, withoutsignificantlyaffecting nascentchain synthesis. By contrast, simple secretory proteins are cotranslationally translocated directly into the ER lumen, without such transient dissociation of translocation from nascent chainelongation. Whileothercomplexsecretoryand membrane proteins have subsequently been found to contain functional PT sequences (8) (R. S. Hegde and V. R. Lingappa, unpublished results), apoB may be unique in the large number of F T sequences that appear to be involved in its biogenesis (6). Recently, progress has been made in understanding the molecular events of one translocational pause of apoB (9). During the translocational pause occurring at apoB-6.7, the tight seal between the ribosome and the translocation channel, which normally serves to shield nascent chains from the cytosol, is dramatically and reversibly opened. At the same time there is a coordinate alteration in the proteins with which the nascent chain associates. As a result, macromolecular interactions can occur between the nascent chain and the cytosol that are not possible during translocation of simple secretoly Abbreviations: apoB, apolipoprotein B; ER, endoplasmic reticulum; MTP, microsomal triglyceride transfer protein; FT, pause transfer; PK, proteinase I(; VLDL, very low density lipoprotein. 'To whom correspondence should be addressed. Journal of Lipid Research Volume 38, 1997 1149 by gest, on N ovem er 6, 2017 w w w .j.org D ow nladed fom proteins. These findings argue strongly that, in at least some cases, translocational pausing is an example of cellular regulation of the translocation machineiy ( 10). In the case of apoB, such regulation seems likely to be related to events of lipoprotein particle formation and secretion or degradation. However, the precise role of this and the other specific PT sequences in apoB biogenesis remains an unsolved problem. It is possible, for example by virtue of differences in flanking sequences, that different PT sequences mediate different types of events in apoB biogenesis. These events may have in common only that they manifest by the available assays as translocational pausing. Thus, only a subset of PT sequences may actually open the ribosome-membrane junction in the manner recently described for the apoB6.7 PT sequence (9). Furthermore, regardless of the mechanism of action of any particular PT sequence, a variety of consequences relevant to lipoprotein particle assembly or secretion might ensue, or be facilitated, during a translocational pause. Exactly which of several possible consequences actually occurs during a particular translocational pause might depend not only on the particular pause in question but also on factors involved in regulation of lipoprotein particle formation (e.g., levels of substrates, metabolites, hormones). A first step in understanding the role of translocational pauses in apoB biogenesis would be achieved by exhaustively mapping the distribution of PT sequences throughout the entire length of apoB-100. Here we report a systematic survey designed to identify all ot the functional PT sequences in apoB-100 and the correlation of their location to proposed structural and functional domains of apoB. We find 23 distinct PT sequences clustered in three regions ofapoB: the N-terminal20% which has nine distinct PT sequences; the apoB48 junction, in whose proximity four PT sequences are found; and apoB-65-95 where ten additional PT sequences are located. Conversely, two large regions, apoB20-44 and apoB-47-67, each encompassing approximately 100 kD or more of the apoB coding region, appear devoid of functional PT sequences. These findings have implications for the relationship of PT sequences to structural features and functional organization of apoB in lipoprotein particles and are discussed