The insertase YidC chaperones the polytopic membrane protein MelB inserting and folding simultaneously from both termini.

The insertion and folding of proteins into membranes is crucial for cell viability. Yet, the detailed contributions of insertases remain elusive. Here, we monitor how the insertase YidC guides the folding of the polytopic melibiose permease MelB into membranes. In vivo experiments using conditionally depleted E. coli strains show that MelB can insert in the absence of SecYEG if YidC resides in the cytoplasmic membrane. In vitro single-molecule force spectroscopy reveals that the MelB substrate itself forms two folding cores from which structural segments insert stepwise into the membrane. However, misfolding dominates, particularly in structural regions that interface the pseudo-symmetric α-helical domains of MelB. Here, YidC takes an important role in accelerating and chaperoning the stepwise insertion and folding process of both MelB folding cores. Our findings reveal a great flexibility of the chaperoning and insertase activity of YidC in the multifaceted folding processes of complex polytopic membrane proteins.

Monitoring the binding and insertion of a single transmembrane protein by an insertase.

Cells employ highly conserved families of insertases and translocases to insert and fold proteins into membranes. How insertases insert and fold membrane proteins is not fully known. To investigate how the bacterial insertase YidC facilitates this process, we here combine single-molecule force spectroscopy and fluorescence spectroscopy approaches, and molecular dynamics simulations. We observe that within 2 ms, the cytoplasmic α-helical hairpin of YidC binds the polypeptide of the membrane protein Pf3 at high conformational variability and kinetic stability. Within 52 ms, YidC strengthens its binding to the substrate and uses the cytoplasmic α-helical hairpin domain and hydrophilic groove to transfer Pf3 to the membrane-inserted, folded state. In this inserted state, Pf3 exposes low conformational variability such as typical for transmembrane α-helical proteins. The presence of YidC homologues in all domains of life gives our mechanistic insight into insertase-mediated membrane protein binding and insertion general relevance for membrane protein biogenesis.

The T7 ejection nanomachine components gp15-gp16 form a spiral ring complex that binds DNA and a lipid membrane.

Bacteriophage T7 initiates infection by ejecting several internal capsid proteins into the host cell; these proteins then assemble into a nanomachine that translocates the viral genome from the phage head into the cytoplasm. The ejected proteins are thought to partially unfold as they pass through the lumen of the portal and the short stubby T7 tail during their entry into the cell. In vivo, the internal proteins gp15 and gp16 assemble into a tubular structure that spans the periplasm and cytoplasmic membrane. We show here that purified gp15 and gp16 can refold from a partially denatured state in vitro, and that gp15 interacts with gp16 to form a spiral ring structure. Purified gp15 binds to DNA, whereas gp16 binds protein-free liposomes; the gp15-gp16 complex binds both DNA and liposomes. Limited proteolysis of the liposome-bound gp16 reveals that its C-terminal region is protected, suggesting a partial membrane insertion of the protein.

Cysteine residues in the transmembrane regions of M13 procoat protein suggest that oligomeric coat proteins assemble onto phage progeny.

The M13 phage assembles in the inner membrane of Escherichia coli. During maturation, about 2,700 copies of the major coat protein move from the membrane onto a single-stranded phage DNA molecule that extrudes out of the cell. The major coat protein is synthesized as a precursor, termed procoat protein, and inserts into the membrane via a Sec-independent pathway. It is processed by a leader peptidase from its leader (signal) peptide before it is assembled onto the phage DNA. The transmembrane regions of the procoat protein play an important role in all these processes. Using cysteine mutants with mutations in the transmembrane regions of the procoat and coat proteins, we investigated which of the residues are involved in multimer formation, interaction with the leader peptidase, and formation of M13 progeny particles. We found that most single cysteine residues do not interfere with the membrane insertion, processing, and assembly of the phage. Treatment of the cells with copper phenanthroline showed that the cysteine residues were readily engaged in dimer and multimer formation. This suggests that the coat proteins assemble into multimers before they proceed onto the nascent phage particles. In addition, we found that when a cysteine is located in the leader peptide at the -6 position, processing of the mutant procoat protein and of other exported proteins is affected. This inhibition of the leader peptidase results in death of the cell and shows that there are distinct amino acid residues in the M13 procoat protein involved at specific steps of the phage assembly process.

Different regions of the nonconserved large periplasmic domain of Escherichia coli YidC are involved in the SecF interaction and membrane insertase activity.

The YidC protein of Escherichia coli is required for inserting Sec-independent membrane proteins and has a supportive role for the insertion of Sec-dependent proteins into the membrane bilayer. Because a portion of YidC copurifies with the Sec translocase, this interaction might be necessary to assist in the membrane insertion of Sec-dependent proteins. This study describes a deletion analysis that investigates which parts of YidC are required for its interaction with the SecDF complex of the Sec translocase and for the function of YidC as an insertase for the Sec-dependent membrane proteins. The results suggest that the first periplasmic region, which includes residues 24-346, is required for the interaction of YidC with the Sec translocase, in particular with the SecF protein. Further studies showed that residues 215-265 of YidC are sufficient for SecF binding. Surprisingly, the interaction of YidC with SecF is not critical for cell viability as YidC, lacking residues 24-264, was fully functional to support the growth of E. coli. It was also observed that this YidC mutant was fully functional to insert the Sec-dependent subunit A of the F(1)F(o) ATP synthase and an M13 procoat derivative, as well as the Sec-independent M13 procoat protein and subunit C of the ATP synthase. Only when additional residues of the periplasmic region were deleted (265-346) was the membrane insertase function of YidC inhibited.