Interaction between glycolipid MPIase and proteinaceous factors during protein integration into the cytoplasmic membrane of .

Protein integration into biomembranes is an essential biological phenomenon common to all organisms. While various factors involved in protein integration, such as SRP, SecYEG and YidC, are proteinaceous, we identified a glycolipid named MPIase (Membrane Protein Integrase), which is present in the cytoplasmic membrane of . experiments using inverted membrane vesicles prepared from MPIase-depleted strains, and liposomes containing MPIase showed that MPIase is required for insertion of a subset of membrane proteins, which has been thought to be SecYEG-independent and YidC-dependent.

Interplay between MPIase, YidC, and PMF during Sec-independent insertion of membrane proteins.

Integral membrane proteins with the N-out topology are inserted into membranes usually in YidC- and PMF-dependent manners. The molecular basis of the various dependencies on insertion factors is not fully understood. A model protein, Pf3-Lep, is inserted independently of both YidC and PMF, whereas the V15D mutant requires both YidC and PMF in vivo.

Assay for Bacterial Membrane Protein Integration into Proteoliposomes.

It is important to experimentally determine how membrane proteins are integrated into biomembranes to unveil the roles of the integration factors, and to understand the functions and structures of membrane proteins. We have developed a reconstitution system for membrane protein integration in using purified factors, in which the integration reaction is highly reproducible. This system enabled not only analysis of membrane-embedded factors including glycolipid MPIase, but also elucidation of the detailed mechanisms underlying membrane protein integration.

Ring assembly of c subunits of F F -ATP synthase in Propionigenium modestum requires YidC and UncI following MPIase-dependent membrane insertion.

The c subunits of F F -ATP synthase (F c) assemble into a ring structure, following membrane insertion that is dependent on both glycolipid MPIase and protein YidC. We analyzed the insertion and assembly processes of Propionigenium modestum F c (Pm-F c), of which the ring structure is resistant to SDS. Ring assembly of Pm-F c requires P.

The bacterial protein YidC accelerates MPIase-dependent integration of membrane proteins.

Bacterial membrane proteins are integrated into membranes through the concerted activities of a series of integration factors, including membrane protein integrase (MPIase). However, how MPIase activity is complemented by other integration factors during membrane protein integration is incompletely understood. Here, using inverted inner-membrane vesicle and reconstituted (proteo)liposome preparations from cells, along with membrane protein integration assays and the PURE system to produce membrane proteins, we found that anti-MPIase IgG inhibits the integration of both the Sec-independent substrate 3L-Pf3 coat and the Sec-dependent substrate MtlA into membrane vesicles.

Biosynthesis of glycolipid MPIase (membrane protein integrase) is independent of the genes for ECA (enterobacterial common antigen).

MPIase (membrane protein integrase) is an essential glycolipid that drives protein integration into the inner membrane of E. coli, while glycolipid ECA (enterobacterial common antigen) is a major component at the surface of the outer membrane. Irrespective of the differences in molecular weight, subcellular localization and function in cells, the glycan chains of the two glycolipids are similar, since the repeating unit comprising the glycan chains is the same.

CdsA is involved in biosynthesis of glycolipid MPIase essential for membrane protein integration in vivo.

MPIase is a glycolipid that is involved in membrane protein integration. Despite evaluation of its functions in vitro, the lack of information on MPIase biosynthesis hampered verification of its involvement in vivo. In this study, we found that depletion of CdsA, a CDP-diacylglycerol synthase, caused not only a defect in phospholipid biosynthesis but also MPIase depletion with accumulation of the precursors of both membrane protein M13 coat protein and secretory protein OmpA.

Membrane insertion of F c subunit of FF ATPase depends on glycolipozyme MPIase and is stimulated by YidC.

The F c subunit of FF ATPase (F-c) possesses two membrane-spanning stretches with N- and C-termini exposed to the periplasmic (extracellular) side of the cytoplasmic membrane of E. coli. Although F-c insertion has been extensively analyzed in vitro by means of protease protection assaying, it is unclear whether such assays allow elucidation of the insertion process faithfully, since the membrane-protected fragment, an index of membrane insertion, is a full-length polypeptide of F-c, which is the same as the protease-resistant conformation without membrane insertion.