Distant downstream sequence determinants can control N-tail translocation during protein insertion into the endoplasmic reticulum membrane.
We have studied the membrane insertion of ProW, an Escherichia coli inner membrane protein with seven transmembrane segments and a large periplasmic N-terminal tail, into endoplasmic reticulum (ER)-derived dog pancreas microsomes. Strikingly, significant levels of N-tail translocation is seen only when a minimum of four of the transmembrane segments are present; for constructs with fewer transmembrane segments, the N-tail remains mostly nontranslocated and the majority of the molecules adopt an "inverted" topology where normally nontranslocated parts are translocated and vice versa. N-tail translocation can also be promoted by shortening of the N-tail and by the addition of positively charged residues immediately downstream of the first trasnmembrane segment. We conclude that as many as four consecutive transmembrane segments may be collectively involved in determining membrane protein topology in the ER and that the effects of downstream sequence determinants may vary depending on the size and charge of the N-tail. We also provide evidence to suggest that the ProW N-tail is translocated across the ER membrane in a C-to-N-terminal direction.
Nilsson
I
I
Department of Biochemistry, Stockholm University, S-10691 Stockholm, Sweden.
Witt
S
S
Kiefer
H
H
Mingarro
I
I
von Heijne
G
G
D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002415:Cats; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins; D006031:Glycosylation; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008861:Microsomes / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D010179:Pancreas / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; C490210:ProW protein, E coli