Essential Function of Protein 4.1G in Targeting of Membrane Protein Palmitoylated 6 into Schmidt-Lanterman Incisures in Myelinated Nerves

Abstract

Protein 4.1G is a membrane skeletal protein found in specific subcellular structures in myelinated Schwann cells and seminiferous tubules. Here, we show that in the mouse sciatic nerve, protein 4.1G colocalized at Schmidt-Lanterman incisures (SLI) and the paranodes with a member of the membrane-associated guanylate kinase (MAGUK) family, membrane protein palmitoylated 6 (MPP6). Coimmunoprecipitation experiments revealed that MPP6 was interacting with protein 4.1G. In contrast to wild-type nerves, in 4.1G knockout mice, MPP6 was found largely in the cytoplasm near Schwann cell nuclei, indicating an abnormal protein transport. Although the SLI remained in the 4.1G knockout sciatic nerves, as confirmed by E-cadherin immunostaining, their shape was altered in aged 4.1G knockout nerves compared to their shape in wild-type nerves. In the seminiferous tubules, MPP6 was localized similarly to protein 4.1G along cell membranes of the spermatogonium and early spermatocytes. However, in contrast to myelinated peripheral nerves, the specific localization of MPP6 in the seminiferous tubules was unaltered in the absence of protein 4.1G. These results indicate that 4.1G has a specific role in the targeting of MPP6 to the SLI and the assembly of these subcellular structures. P rotein 4.1G (4.1G) is a member of the 4.1 family (27, 46), a group of membrane skeletal proteins that link various components to the spectrin-actin network (10). We have previously reported that 4.1G is present in rodent Schwann cells (7, 24) and mouse seminiferous tubules (40, 41). In peripheral nerves, 4.1G is found at the Schmidt-Lanterman incisures (SLI) and the paranodal loops of myelinated Schwann cells (24). The SLI are funnelshaped interruptions within the myelin sheath of nerve fibers. They contain high concentrations of actin and spectrin (35, 42), which forms a membrane skeleton that might contribute to the elasticity and stability of these structures.Membrane-associated guanylate kinase (MAGUK) family proteins contain PDZ (for postsynaptic density 95 [PSD-95]/Drosophila disks large [Dlg]/zonula occludens 1 [ZO-1]), GUK (guanylate kinase), and SH3 (src homology 3) domains, and they localize to specific domains at the plasma membranes (9, 11). In epithelial cells, for example, some MAGUKs, such as Dlg and ZO-1, are required for the formation of adherens and tight junctions, respectively. In addition to their function as membrane scaffolds (16, 21), several MAGUKs also control intracellular protein transport through their ability to bind motor proteins (45, 49). Interestingly, some MAGUKs contain specific domains that interact with 4.1 proteins (14,17,18). In the current study, we report the identification of membrane protein palmitoylated 6 (MPP6) (also known as PALS2, VAM1, and p55T [http://www .genenames.org/]) as a novel MAGUK molecule that interacts with 4.1G in peripheral nerves. We further demonstrate that the interaction between 4.1G and MPP6 is essential for the targeting of the latter into SLI. MATERIALS AND METHODS Animals and anesthes...

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