Lipid-protein interactions drive membrane protein topogenesis in accordance with the positive inside rule

Abstract

Transmembrane domain orientation within some membrane proteins is dependent on membrane lipid composition. Initial orientation occurs within the translocon, but final orientation is determined after membrane insertion by interactions within the protein and between lipid headgroups and protein extramembrane domains. Positively and negatively charged amino acids in extramembrane domains represent cytoplasmic retention and membrane translocation forces, respectively, which are determinants of protein orientation. Lipids with no net charge dampen the translocation potential of negative residues working in opposition to cytoplasmic retention of positive residues, thus allowing the functional presence of negative residues in cytoplasmic domains without affecting protein topology.

FIGURE 1.

Pathway for synthesis of native and foreign lipids in E. coli. The following enzymes are indicated with their respective genes (from E. coli (17) unless indicated otherwise): 1, CDP-diacylglycerol synthase; 2, phosphatidylserine synthase; 3, phosphatidylserine decarboxylase; 4, PGP synthase; 5, PGP phosphatase; 6, CL synthase; 7, PG, pre-MDO (membrane-derived oligosaccharide) sn-glycerol-1-P transferase; 8, diacylglycerol kinase; 9, MGlcDAG synthase (Acholeplasma laidlawii) (19); 10, DGlcDAG synthase (A. laidlawii) (20); 11, PC synthase (Legionella pneumophila) (29). The X in phosphatidic acid is an OH and is in the position that changes depending on the downstream pathway. The lipids in black (∼5% of total phospholipids) and red are native to E. coli. The lipids in green are foreign lipids introduced into E. coli carrying the indicated genes.

FIGURE 2.

Topological organization of secondary transporters as a function of membrane lipid composition. The cytoplasm is at the top of each figure; TMs are noted by rectangles; NT and CT refer to the N and C termini, respectively; and extramembrane domains oriented to the cytoplasm (C) or periplasm (P) in PE-containing cells are indicated. A, LacY topology as determined in PE-containing (+PE) cells is depicted. Green (negative charge) and red (positive charge) dots indicate the approximate positions of charged residues with the net charge of each extramembrane domain noted next to the domain name. B, LacY topology as determined in PE-lacking cells is shown. The exposure of TMVII (red) to the periplasm results in the loss of salt bridges with TMX and TMXI. C, shown is LacY topology determined after induction of PE synthesis in cells where assembly of LacY initially occurred in the absence of PE. D, PheP and GabP topology (topology of the lipid-insensitive TMV to CT domains not shown) as determined in PE-containing cells is shown. The same nomenclature is used as in A. E, the topology of PheP and GabP assembled in cells lacking PE is shown. The high content of aromatic residues in TMIII is indicated.

FIGURE 3.

PE and the positive inside rule. A cytoplasmic domain containing both negatively and positively charged amino acids is shown. PE dampens the translocation potential of negative residues in favor of the cytoplasmic retention potential of positive residues (left). The effective net positive charge (+3) of the domain is sufficient for cytoplasmic retention. Without PE, the net positive charge is + 1, which is insufficient for cytoplasmic retention with the possibility that negative residues are dominant over positive residues in the absence of PE (right). The protonmotive force (arrow) positive outward determines domain directional movement depending on the domain effective net charge as influenced by the lipid environment. Induction of PE synthesis post-assembly of a protein reverses the effective net charge and drives TM flipping (8).