IDENTIFICATION OF AN INTERNAL TOPOGENIC SIGNAL SEQUENCE IN HUMAN BAND-3, THE ERYTHROCYTE ANION-EXCHANGER

The insertion of Band 3, the human erythrocyte anion exchanger, into m icrosomal membranes was studied in an in vitro reticulocyte lysate tra nslation system. Band 3 consists of a 43-kDa amino-terminal cytosolic domain and a carboxyl-terminal 52-kDa membrane domain containing up to 14 transmembrane segments with a single N-glycosylation site at Asn-6 42. Insertion of truncated Band 3 molecules into microsomal membranes was assayed by glycosylation, resistance to alkaline extraction, and t ryptic removal of the cytosolic domain. Truncations containing either the first four or the last eight putative transmembrane segments were stably integrated into microsomes showing that an intact membrane doma in was not required for membrane integration. Furthermore, the extracy tosolic domain following the seventh transmembrane segment was properl y translocated across the microsomal membrane and glycosylated whether the seventh transmembrane segment was the first, last, or the only tr ansmembrane segment in the construct. The ability of the entire membra ne domain, the truncated domain beginning with the seventh transmembra ne segment, or the seventh transmembrane segment to insert into micros omes was dependent on the presence of the signal recognition particle receptor. The seventh transmembrane segment in Band 3 therefore has th e topogenic properties of an internal signal sequence.