Structures and Molecular Composition of Schmidt-Lanterman Incisures.

Schmidt-Lanterman incisure (SLI) is a circular-truncated cone shape in the myelin internode that is a specific feature of myelinated nerve fibers formed in Schwann cells in the peripheral nervous system (PNS). The SLI circular-truncated cones elongate like spring at the narrow sites of beaded appearance nerve fibers under the stretched condition. In this chapter, we demonstrate various molecular complexes in SLI, and especially focus on membrane skeleton, protein 4.1G-membrane protein palmitoylated 6 (MPP6)-cell adhesion molecule 4 (CADM4). 4.1G was essential for the molecular targeting of MPP6 and CADM4 in SLI. Motor activity and myelin ultrastructures were abnormal in 4.1G-deficient mice, indicating the 4.1G function as a signal for proper formation of myelin in PNS. Thus, SLI probably has potential roles in the regulation of adhesion and signal transduction as well as in structural stability in Schwann cell myelin formation.

An allosteric link connecting the lipid-protein interface to the gating of the nicotinic acetylcholine receptor.

The mechanisms underlying lipid-sensing by membrane proteins is of considerable biological importance. A unifying mechanistic question is how a change in structure at the lipid-protein interface is translated through the transmembrane domain to influence structures critical to protein function. Gating of the nicotinic acetylcholine receptor (nAChR) is sensitive to its lipid environment. To understand how changes at the lipid-protein interface influence gating, we examined how a mutation at position 418 on the lipid-facing surface of the outer most M4 transmembrane α-helix alters the energetic couplings between M4 and the remainder of the transmembrane domain. Human muscle nAChR is sensitive to mutations at position 418, with the Cys-to-Trp mutation resulting in a 16-fold potentiation in function that leads to a congenital myasthenic syndrome. Energetic coupling between M4 and the Cys-loop, a key structure implicated in gating, do not change with C418W. Instead, Trp418 and an adjacent residue couple energetically with residues on the M1 transmembrane α-helix, leading to a reorientation of M1 that stabilizes the open state. We thus identify an allosteric link connecting the lipid-protein interface of the nAChR to altered channel function.

Lipid anchoring of Arabidopsis phototropin 1 to assess the functional significance of receptor internalization: should I stay or should I go?

The phototropin 1 (phot1) blue light receptor mediates a number of adaptive responses, including phototropism, that generally serve to optimize photosynthetic capacity. Phot1 is a plasma membrane-associated protein, but upon irradiation, a fraction is internalized into the cytoplasm. Although this phenomenon has been reported for more than a decade, its biological significance remains elusive. Here, we use a genetic approach to revisit the prevalent hypotheses regarding the functional importance of receptor internalization. Transgenic plants expressing lipidated versions of phot1 that are permanently anchored to the plasma membrane were used to analyse the effect of internalization on receptor turnover, phototropism and other phot1-mediated responses. Myristoylation and farnesylation effectively prevented phot1 internalization. Both modified photoreceptors were found to be fully functional in Arabidopsis, rescuing phototropism and all other phot1-mediated responses tested. Light-mediated phot1 turnover occurred as in the native receptor. Furthermore, our work does not provide any evidence of a role of phot1 internalization in the attenuation of receptor signalling during phototropism. Our results demonstrate that phot1 signalling is initiated at the plasma membrane. They furthermore indicate that release of phot1 into the cytosol is not linked to receptor turnover or desensitization.

[Mycoplasma genitalium lipid-associated membrane proteins induce endocervical epithelial cells to express ß-defensin-2].

OBJECTIVE: To observe the expression of human ß-defensin-2 (hBD-2) induced by Mycoplasma genitalium-derived lipid-associated membrane proteins (LAMPs) and its potential mechanism. METHODS: Human endocervical epithelial End1/E6E7 cells were cultured in vitro and stimulated by different concentrations of LAMPs for 48 hours, and the expressions of hBD-2 mRNA and protein were detected by real-time RT-PCR and Western blotting, respectively. Toll-like receptor 2 (TLR2) and TLR6 neutralizing antibodies for End1/E6E7 cell cultivation, and dominant negative plasmids for cell transfection were used to analyze the roles of TLR2, TLR6 and MyD88 in mediating hBD-2 expression. Nuclear translocation of the nuclear factor κB (NF-κB) p65 subunit, and its DNA-binding activity were detected by Western blotting and electrophoretic mobility shift assay (EMSA), respectively. Pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB, was used to investigate the effect of NF-κB on hBD-2 expression. RESULTS: Mycoplasma genitalium-derived LAMPs induced the expressions of hBD-2 mRNA and protein in End1/E6E7 cells. The expressions could be abrogated by TLR2 and TLR6 neutralizing antibodies, or their dominant negative plasmids. In addition, dominant negative plasmids of MyD88 significantly decreased LAMPs-induced hBD-2 expression. Western blotting showed that p65 was translocated to the nucleus, and the DNA binding activity of NF-κB was enhanced after LAMPs treatment. Furthermore, PDTC treatment decreased LAMPs- induced hBD-2 expression. CONCLUSION: Mycoplasma genitalium-derived LAMPs can induce End1/E6E7 cells to express hBD-2, which may be involved in the TLR2, TLR6/Myd88/NF-κB pathways.

Activation of the Escherichia coli ß-barrel assembly machine (Bam) is required for essential components to interact properly with substrate.

The outer membrane (OM) of gram-negative bacteria such as Escherichia coli contains lipoproteins and integral ß-barrel proteins (outer-membrane proteins, OMPs) assembled into an asymmetrical lipid bilayer. Insertion of ß-barrel proteins into the OM is mediated by a protein complex that contains the OMP BamA and four associated lipoproteins (BamBCDE). The mechanism by which the Bam complex catalyzes the assembly of OMPs is not known. We report here the isolation and characterization of a temperature-sensitive lethal mutation, bamAE373K, which alters the fifth polypeptide transport-associated domain and disrupts the interaction between the BamAB and BamCDE subcomplexes. Suppressor mutations that map to codon 197 in bamD restore Bam complex function to wild-type levels. However, these suppressors do not restore the interaction between BamA and BamD; rather, they bypass the requirement for stable holocomplex formation by activating BamD. These results imply that BamA and BamD interact directly with OMP substrates.

A lipid-anchored SNARE supports membrane fusion.

Intracellular membrane fusion requires R-SNAREs and Q-SNAREs to assemble into a four-helical parallel coiled-coil, with their hydrophobic anchors spanning the two apposed membranes. Based on the fusion properties of chemically defined SNARE- proteoliposomes, it has been proposed that the assembly of this helical bundle transduces force through the entire bilayer via the transmembrane SNARE anchor domains to drive fusion. However, an R-SNARE, Nyv1p, with a genetically engineered lipid anchor that spans half of the bilayer suffices for the fusion of isolated vacuoles, although this organelle has other R-SNAREs. To demonstrate unequivocally the fusion activity of lipid-anchored Nyv1p, we reconstituted proteoliposomes with purified lipid-anchored Nyv1p as the only protein. When these proteoliposomes were incubated with those bearing cognate Q-SNAREs, there was trans-SNARE complex assembly but, in accord with prior studies of the neuronal SNAREs, little lipid mixing. However, the addition of physiological fusion accessory proteins (HOPS, Sec17p, and Sec18p) allows lipid-anchored Nyv1p to support fusion, suggesting that trans-SNARE complex function is not limited to force transduction across the bilayers through the transmembrane domains.