INSERTION OF THE POLYTOPIC MEMBRANE-PROTEIN LACTOSE PERMEASE OCCURS BY MULTIPLE MECHANISMS

Citation
Kh. Zen et al., INSERTION OF THE POLYTOPIC MEMBRANE-PROTEIN LACTOSE PERMEASE OCCURS BY MULTIPLE MECHANISMS, Biochemistry, 34(10), 1995, pp. 3430-3437
Citations number
43
Categorie Soggetti
Biology
Journal title
ISSN journal
00062960
Volume
34
Issue
10
Year of publication
1995
Pages
3430 - 3437
Database
ISI
SICI code
0006-2960(1995)34:10<3430:IOTPML>2.0.ZU;2-F
Abstract
The lactose permease of Escherichia coli has 12 transmembrane hydropho bic domains in probable and-helical conformation connected by hydrophi lic loops. Previous studies [Consler, T. G., Persson, B., et al. (1993 ) Proc. Natl. Acad. Sci. U.S.A. 90, 6934-6938] demonstrate that a pept ide fragment (the XB domain) containing a factor Xa protease site imme diately upstream of a biotin acceptor domain can be engineered into th e permease, thereby allowing rapid purification to a high state of pur it:y. Here we describe the use of the XB domain to probe topology and insertion. Cells expressing permease with the XB domain at the N termi nus, at the C terminus, or in loop 6 or 10 on the cytoplasmic face of the membrane catalyze active transport, although only the chimeras wit h the XB domain at the C terminus or in loop 6 are biotinylated. In co ntrast, chimeras with the XB domain in periplasmic loop 3 or 7 are ina ctive, but strikingly, both constructs are biotinylated. Furthermore, the XB domain in all the constructs, particularly in the loop 3 and lo op 7 chimeras, is accessible from the cytoplasmic face of the membrane , as evidenced by factor Xa proteolysis or avidin binding studies with spheroplasts and disrupted membrane preparations. Finally, alkaline p hosphatase fusions one loop downstream from each periplasmic XB domain exhibit high phosphatase activity. Thus, the presence of the XB domai n in a periplasmic loop apparently blocks translocation of a discrete segment of the permease consisting of the loop and the two adjoining h elices without altering insertion of the remainder of the protein. The results provide a strong indication that XB domain insertion cannot b e used to study the topology of polytopic membrane proteins. On the ot her hand, the approach yields unique and important information regardi ng insertion, and it seems likely that certain regions of lactose perm ease may be inserted as helical hairpins. The findings are discussed i n the context of other observations indicating that different regions of the permease may be inserted by different mechanisms.