Diversity and versatility of lipid-protein interactions revealed by molecular genetic approaches

Abstract

The diversity in structures and physical properties of lipids provides a wide variety of possible interactions with proteins that affect their assembly, organization, and function either at the surface of or within membranes. Because lipids have no catalytic activity, it has been challenging to define many of their precise functions in vivo in molecular terms. Those processes responsive to lipids are attuned to the native lipid environment for optimal function, but evidence that lipids with similar properties or even detergents can sometimes partially replace the natural lipid environment has led to uncertainty as to the requirement for specific lipids. The development of strains of microorganisms in which membrane lipid composition can be genetically manipulated in viable cells has provided a set of reagents to probe lipid functions. These mutants have uncovered previously unrecognized roles for lipids and provided in vivo verification for putative functions described in vitro. In this review, we summarize how these reagent strains have provided new insight into the function of lipids. The role of specific lipids in membrane protein folding and topological organization is reviewed. The evidence is summarized for the involvement of anionic lipid-enriched domains in the organization of amphitropic proteins on the membrane surface into molecular machines involved in DNA replication and cell division.

Fig. 1

Biosynthetic pathways for phospholipids in E. coli and CL in eukaryotic cells. The genes encoding the enzyme for each step (1–4, 6 in blue) in E. coli and eukaryotic cells (4A and 6A in yellow) are shown. No gene has been identified for step 5. Note the difference in substrates for CL synthesis shown in steps 6 and 6A for E. coli versus eukaryotic cells, respectively.

Fig. 2

Topological organization of LacY from E. coli in PE-containing and PE-lacking cells. The upper panel shows the packing of LacY into the two separate six TM N-terminal (N) and C-terminal (C) regions with respect to the plane of the membrane bilayer (IN, cytoplasmic side and OUT, periplasmic side). The position of bound substrate is between the two separate domains of the C154G mutant locked in the inwardly open conformation [31]. Reproduced with permission from Science. The center and lower panels are a planar depiction of the nearest neighbor relations and orientation of TMs (color gradient from light to dark in the outward direction for PE-containing cells) of LacY in PE-containing (+PE) and PE-lacking cells (−PE), respectively. The TMs are numbered in Roman numerals consecutively from the N-(NH₂) to C-(COOH) terminus. The cytoplasmic (C, green) and periplasmic (P, blue) extramembrane domains are similarly numbered consecutively as they are oriented in PE-containing cells. The light blue and pink circles indicate the position of residues most important for substrate binding and proton translocation, respectively. The + and − symbols refers to charged residues in the TMs. Note that C6 (light green helix, top of top panel) connects the two separate helical bundles and that P7 is shown with different conformations in PE-containing and PE-lacking cells.

Fig. 3

Scheme for membrane insertion and polymerization–depolymerization of MinD. (A) At the cell pole, cytosolic MinD-ADP (yellow) with its unstructured C-terminal domain (green) exchanges ADP for ATP with an accompanying conformational change in MinD-ATP (blue), resulting in exposure of the C-terminal amphipathic motif (green–gray) followed by electrostatic attraction to the anionic lipid-enriched domain (red phospho-lipid head groups) at the cell pole. Penetration into the phospholipid bilayer is accompanied by amphipathic α-helix formation with alignment parallel to the membrane surface and complementary hydrophobic and ionic interactions. (B) Consecutive binding of MinD-ATP monomers to the membrane as shown in A results in formation of MinD-ATP polymer growing from cell pole to cell center. Near the cell center, binding of MinE (pink) to MinD-ATP induces ATP hydrolysis coupled to a return to the MinD-ADP conformation accompanied by detachment of MinDE. Consecutive detachment of MinD results in depolymerization from the cell center to the cell pole. Cytosolic MinD-ADP diffuses to the opposite cell pole where the cycle is repeated.

Fig. 4

Distribution of MinD and MGlcDAG synthase in wild-type and PE-lacking E. coli. (A) Fluorescent image of YFP-MinD (yellow fluorescent protein) in a living cell (yellow line encircles cell of normal length) with wild-type phospholipid composition (top left) [138]. Schematic representation (bottom left) of coiled MinD polymer structure originating at the cell pole enriched in anionic phospholipids (blue). Fluorescent image of GFP-MinD (top right) in PE-lacking cell (yellow line encircles multinucleated filamentous cell) displayed as distinct focal points [130]. Schematic representation (bottom right) of coiled MinD polymer interacting as concentrated domains in association with multiple anionic phospholipid domains (blue). (B) GFP-MGlcDAG synthase localization at the poles of wild-type cells (left) and as focal points in filamentous PE-lacking cells (right) [100].

Fig. 5

Rejuvenation of DnaA activity by anionic phospholipids and oriC. Multiple DnaA molecules (yellow), bound with either ATP or ADP, bind to oriC near the initiation site on the DNA duplex (blue). Only the DnaA-ATP form (right diagram) is active and capable of opening the DNA duplex as shown to allow assembly of the replisome and initiation of replication. The inactive DnaA-ADP form can be rejuvenated by exchange of ATP for ADP, but this only occurs when DnaA is bound to both oriC and anionic phospholipids shown as red phospholipid head groups organized into an anionic lipid domain on the membrane surface.