Alterations in the Arf6-regulated plasma membrane endosomal recycling pathway in cells overexpressing the tetraspan protein Gas3/PMP22

Abstract

Growth arrest specific 3 (Gas3)/peripheral myelin protein 22 (PMP22) is a component of the compact peripheral nerve myelin, and mutations affecting gas3/PMP22 gene are responsible for a group of peripheral neuropathies in humans. We have performed in vivo imaging in order to investigate in detail the phenotype induced by Gas3/PMP22 overexpression in cultured cells. Here we show that Gas3/PMP22 triggers the accumulation of vacuoles, before the induction of cell death or of changes in cell spreading. Overexpressed Gas3/PMP22 accumulates into two distinct types of intracellular membrane compartments. Gas3/PMP2 accumulates within late endosomes close to the juxtanuclear region, whereas in the proximity of the cell periphery, it induces the formation of actin/phosphatidylinositol (4,5)-bisphosphate(PIP2)-positive large vacuoles. Gas3/PMP22-induced vacuoles do not contain transferrin receptor, but instead they trap membrane proteins that normally traffic through the ADP-ribosylation factor 6 (Arf6) endosomal compartment. Arf6 and Arf6-Q67L co-localize with Gas3/PMP22 in these vacuoles,and the dominant negative mutant of Arf6, T27N, blocks the appearance of vacuoles in response to Gas3/PMP22, but not its accumulation in the late endosomes. Finally a point mutant of Gas3/PMP22 responsible for the Charcot-Marie-Tooth 1A disease is unable to trigger the accumulation of PIP2-positive vacuoles. Altogether these results suggest that increased Gas3/PMP22 levels can alter membrane traffic of the Arf6 plasma-membrane–endosomal recycling pathway and show that, similarly to other tetraspan proteins, Gas3/PMP22 can accumulate in the late endosomes.

Modulation of Epithelial Morphology, Monolayer Permeability, and Cell Migration by Growth Arrest Specific 3/Peripheral Myelin Protein 22

“…In several cell types the overexpression of PMP22 affects cellular morphology (Brancolini et al, 1999;Chies et al, 2003). The altered cell shape observed in PMP22-MDCK monolayers is likely the consequence of reduced cell density at confluence, in which a flattened morphology is necessary for maintaining functional cell-cell junctions.…”

“…In NIH 3T3 fibroblasts, PMP22 overexpression regulates cell spreading, an effect that is dependent on the Rho-GTPase pathway (Brancolini et al, 1999). Recent studies have detected exogenous PMP22 in ADP-ribosylation factor 6 (Arf-6)-positive plasma membrane-endosomal recycling vacuoles before apoptosis or changes in cell shape (Chies et al, 2003). This pathway is known to be involved in modulating the actin cytoskeleton, cell polarity, adhesion, and migration (Donaldson, 2003).…”

“…Transmembrane proteins of the apical junctional complex such as the claudins, occludin, and the junctional adhesion molecules (González-Mariscal et al, 2003) all participate in the regulation of junctional permeability. In addition, based on the findings of Brancolini and colleagues (Brancolini et al, 1999(Brancolini et al, , 2000Chies et al, 2003), PMP22 might be involved in the regulation of epithelial proliferation and/or cell migration, dynamic processes that involve changes in cell adhesion and morphology. In support of this possibility, PMP22 contains the carbohydrate L2/HNK1 adhesion/recognition epitope in the first extracellular loop (Snipes et al, 1993;Schachner et al, 1995).…”

Glycan-independent Role of Calnexin in the Intracellular Retention of Charcot-Marie-Tooth 1A Gas3/PMP22 Mutants

“…Microinjection and Time Lapse-Nuclear microinjection was performed using the Automated Injection System (Zeiss-Germany) as described previously (26). Different expression vectors were injected into the nuclei of cells for 0.5 s at a constant pressure of 50 hectopascals.…”

The TBC (Tre-2/Bub2/Cdc16) Domain Protein TRE17 Regulates Plasma Membrane-Endosomal Trafficking through Activation of Arf6

“…Two additional proteins that appear to regulate this pathway are EHD1 and Gas3/PMP22 (10,15). Overexpression of EHD1 enhances recycling of cargo to the plasma membrane, while overexpression of Gas3/PMP22 induces morphological alterations in the tubular endosome.…”