Membrane insertion and assembly of ductin: a polytopic channel with dual orientations.
Ductin is a highly conserved and polytopic transmembrane protein which is the subunit c component of the vacuolar H(+)-ATPase (V-ATPase) and a component of a connexon channel of gap junctions. Previous studies have suggested that ductin in the V-ATPase has the opposite orientation of ductin in a connexon. Using an in vitro translation system coupled to microsomes derived from the endoplasmic reticulum, we show that ductin is co-translationally inserted into the membrane bilayer, suggesting a dependency on the signal recognition particle for synthesis. By attaching a C-terminal polypeptide derived from beta-lactamase and by using cysteine replacement coupled to chemical labelling, we show that ductin is inserted into the microsomal membrane in both orientations in similar proportions. In contrast, squid rhodopsin appears to be inserted in a single orientation. Changing conserved charged residues at the N-terminus of ductin does not affect the ratio of the two orientations. Once in the microsomal membrane, ductin assembles into an oligomeric complex which contains a pore accessible to a water-soluble probe, reminiscent of the ductin complex found in the V-ATPase and a connexon.
Dunlop
J
J
CRC Beatson Laboratories, Beatson Institute for Cancer Research, Bearsden, Glasgow, UK.
Jones
P C
PC
Finbow
M E
ME
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003545:Cysteine / Q000502:physiology; D049832:Decapodiformes; D005452:Fluoresceins; D007425:Intracellular Membranes / Q000378:metabolism*; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation / Q000502:physiology; D014176:Protein Biosynthesis; D011487:Protein Conformation; D011510:Proteolipids / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D012243:Rhodopsin / Q000378:metabolism; D025262:Vacuolar Proton-Translocating ATPases; D001618:beta-Lactamases / Q000235:genetics
D005452:Fluoresceins; D011510:Proteolipids; D011993:Recombinant Fusion Proteins; C082471:ductin; C037041:fluorescein 5-maleimide; D012243:Rhodopsin; D001618:beta-Lactamases; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases; D003545:Cysteine