The myelin proteolipid plasmolipin forms oligomers and induces liquid-ordered membranes in the Golgi complex.

Myelin comprises a compactly stacked massive surface area of protein-poor thick membrane that insulates axons to allow fast signal propagation. Increasing levels of the myelin protein plasmolipin (PLLP) were correlated with post-natal myelination; however, its function is unknown. Here, the intracellular localization and dynamics of PLLP were characterized in primary glial and cultured cells using fluorescently labeled PLLP and antibodies against PLLP. PLLP localized to and recycled between the plasma membrane and the Golgi complex. In the Golgi complex, PLLP forms oligomers based on fluorescence resonance energy transfer (FRET) analyses. PLLP oligomers blocked Golgi to plasma membrane transport of the secretory protein vesicular stomatitis virus G protein (VSVG), but not of a VSVG mutant with an elongated transmembrane domain. Laurdan staining analysis showed that this block is associated with PLLP-induced proliferation of liquid-ordered membranes. These findings show the capacity of PLLP to assemble potential myelin membrane precursor domains at the Golgi complex through its oligomerization and ability to attract liquid-ordered lipids. These data support a model in which PLLP functions in myelin biogenesis through organization of myelin liquid-ordered membranes in the Golgi complex.

The MARVEL transmembrane motif of occludin mediates oligomerization and targeting to the basolateral surface in epithelia.

Occludin (Ocln), a MARVEL-motif-containing protein, is found in all tight junctions. MARVEL motifs are comprised of four transmembrane helices associated with the localization to or formation of diverse membrane subdomains by interacting with the proximal lipid environment. The functions of the Ocln MARVEL motif are unknown. Bioinformatics sequence- and structure-based analyses demonstrated that the MARVEL domain of Ocln family proteins has distinct evolutionarily conserved sequence features that are consistent with its basolateral membrane localization. Live-cell microscopy, fluorescence resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC) were used to analyze the intracellular distribution and self-association of fluorescent-protein-tagged full-length human Ocln or the Ocln MARVEL motif excluding the cytosolic C- and N-termini (amino acids 60-269, FP-MARVEL-Ocln). FP-MARVEL-Ocln efficiently arrived at the plasma membrane (PM) and was sorted to the basolateral PM in filter-grown polarized MDCK cells. A series of conserved aromatic amino acids within the MARVEL domain were found to be associated with Ocln dimerization using BiFC. FP-MARVEL-Ocln inhibited membrane pore growth during Triton-X-100-induced solubilization and was shown to increase the membrane-ordered state using Laurdan, a lipid dye. These data demonstrate that the Ocln MARVEL domain mediates self-association and correct sorting to the basolateral membrane.

The length of cargo-protein transmembrane segments drives secretory transport by facilitating cargo concentration in export domains.

The cellular destination of secretory proteins is determined by interactions of their targeting motifs with coat-protein complexes. The transmembrane domain (TMD) of secretory proteins also plays a central role in their transport and targeting. However, a comprehensive model that considers both TMD- and targeting-sequence-mediated transport has never been advanced. We focused on the secretory transport of two fluorescently tagged membrane proteins: vesicular stomatitis virus G tsO45 (VSVG), which is a cargo protein that is a thermoreversible mutant, and the Golgi-resident protein GalT-CFP. A quantitative approach was applied to analyze, in living cells, secretory transport dynamics, as well as cargo concentration of YFP-tagged VSVG mutants with one, three, five, seven, eight or nine amino acids deleted from their TMD, as well as two or four amino acids added to their TMD. Changes in TMD length affected secretory transport dynamics and the extent of cargo concentration in the ER exit sites, demonstrating that the capacity of the transport machinery to concentrate cargo depends on the length of the TMD of the cargo protein.