Alterations in the Arf6-regulated plasma membrane endosomal recycling pathway in cells overexpressing the tetraspan protein Gas3/PMP22.
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 (PIP(2))-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 PIP(2)-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.
Chies
Romina
R
Dipartimento di Scienze e Tecnologie Biomediche, Sezione di Biologia, Universita' di Udine, P.le Kolbe 4, 33100 Udine, Italy.
Nobbio
Lucilla
L
Edomi
Paolo
P
Schenone
Angelo
A
Schneider
Claudio
C
Brancolini
Claudio
C
D016475:3T3 Cells; D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000378:metabolism*; D000199:Actins / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism*; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins / Q000235:genetics; D008246:Lysophospholipids / Q000378:metabolism; D051379:Mice; D050178:Monoglycerides; D009185:Myelin Proteins / Q000235:genetics* / Q000378:metabolism; D010523:Peripheral Nervous System Diseases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D021381:Protein Transport / Q000502:physiology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012583:Schwann Cells / Q000166:cytology; D012584:Sciatic Nerve / Q000166:cytology; D014617:Vacuoles / Q000378:metabolism; D054460:rho-Associated Kinases
D000089982:ADP-Ribosylation Factor 6; D000199:Actins; D007202:Indicators and Reagents; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D008246:Lysophospholipids; D050178:Monoglycerides; D009185:Myelin Proteins; C489545:Pmp22 protein, mouse; C489546:Pmp22 protein, rat; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins; C012786:bis(monoacylglyceryl)phosphate; D049452:Green Fluorescent Proteins; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human; C000715727:Arf6 protein, mouse; C000715349:Arf6 protein, rat