Alterations in the Arf6-regulated plasma membrane endosomal recycling pathway in cells overexpressing the tetraspan protein Gas3/PMP22
Alterations in the Arf6-regulated plasma membrane endosomal recycling pathway in cells overexpressing the tetraspan protein Gas3/PMP22
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DOI：
10.1242/jcs.00326
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发表时间：
2003-03-15
影响因子：
4
通讯作者：
Brancolini, C
中科院分区：
文献类型：
--
作者：
Chies, R;Nobbio, L;Brancolini, C
生长停滞特异性3(Gas3)/外周髓鞘蛋白22(PMP22)是致密的周围神经髓鞘的组成部分，影响Gas3/PMP22基因的突变与人类一组周围神经病有关。我们进行了体内成像，以详细研究Gas3/PMP22过表达在培养细胞中诱导的表型。在这里，我们证明了Gas3/PMP22在诱导细胞死亡或细胞伸展变化之前，触发了空泡的积累。过表达的Gas3/PMP22聚集成两种不同类型的细胞内膜。Gas3/PMP2聚集在靠近核旁区域的晚期内吞体内，而在靠近细胞周边的区域，它诱导形成肌动蛋白/磷脂酰肌醇(4，5)-二磷酸(PIP2)阳性的大空泡。Gas3/PMP22诱导的空泡不包含转铁蛋白受体，而是捕获通常通过ADP-核糖化因子6(Arf6)内糖化间隔运输的膜蛋白。Arf6和Arf6-Q67L与Gas3/PMP22共定位于这些液泡中，Arf6的显性负性突变体T27N阻止了对Gas3/PMP22反应的空泡的出现，但不能阻止其在晚期内体中的积累。最后，导致Charcot-Marie-Tooth 1A病的Gas3/PMP22的一个点突变不能触发PIP2阳性空泡的积累。总之，这些结果表明，Gas3/PMP22水平的增加可以改变Arf6质膜-膜-内体循环途径的膜运输量，并表明，与其他Tetraspan蛋白类似，Gas3/PMP22可以在晚期内体中积聚。
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.