Intracellular retention of glycosylphosphatidyl inositol-linked proteins in caveolin-deficient cells
Intracellular retention of glycosylphosphatidyl inositol-linked proteins in caveolin-deficient cells
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DOI：
10.1128/mcb.22.11.3905-3926.2002
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发表时间：
2002-06-01
影响因子：
5.3
通讯作者：
Lisanti, MP
中科院分区：
文献类型：
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作者：
Sotgia, F;Razani, B;Lisanti, MP
糖基磷脂酰肌醇 (GPI) 连接蛋白和小窝蛋白之间的关系仍然存在争议。在这里，我们从 Cav-1 无效小鼠胚胎中衍生出成纤维细胞，以研究在没有小窝蛋白的情况下 GPI 连接蛋白的行为。这些细胞缺乏形态学上的小凹，不表达小窝蛋白 1，并且小窝蛋白 2 表达显示出类似 95% 的下调；这些细胞也不表达 Caveolin-3（一种肌肉特异性的 Caveolin 家族成员）。因此，这些小窝蛋白缺陷细胞代表了研究小窝蛋白在 GPI 相关蛋白质分选中的作用的理想工具。我们发现，在 Cav-1 无效细胞中，GPI 连接蛋白优先保留在细胞内隔室中，我们将其称为高尔基复合体。这种细胞内的 GPI 连接蛋白库不会被降解，并且根据其 Triton 不溶性判断，仍然与细胞内脂筏相关。相反，正如预期的那样，GPI 连接蛋白在野生型细胞中被转运至质膜。此外，Caveolin-1 或 Caveolin-3（但不是 Caveolin-2）在 Cav-1 无效细胞中的重组表达补充了这种表型，并恢复了 GPI 连接蛋白的细胞表面表达。这也许令人惊讶，因为 GPI 连接蛋白仅限于膜的外质小叶，而小窝蛋白是细胞质定向的膜蛋白。由于 Caveolin-1 通常在三个半胱氨酸残基（133、143 和 156）上进行棕榈酰化，因此我们推测棕榈酰化可能以机械方式将 Caveolin-1 与 GPI 连接蛋白偶联。为了支持这一假设，我们表明，在该互补测定中，恢复 GPI 连接蛋白的细胞表面表达需要 Caveolin-1 在残基 143 和 156（而非残基 133）上的棕榈酰化。我们还表明，另一种脂筏相关蛋白 c-Src 保留在 Cav-1 无效细胞的细胞内。因此，高尔基体相关的小窝蛋白和小窝样囊泡可能代表了有效地将脂筏及其相关蛋白质从反高尔基体转移到质膜所必需的运输机制的一部分。为了进一步支持这些发现，GPI 连接蛋白也保留在来自 Cav-1 缺失小鼠（即肺内皮细胞和肾上皮细胞）和 Cav-3 缺失小鼠（骨骼肌纤维）的组织样本中的细胞内。
The relationship between glycosylphosphatidyl inositol (GPI)-linked proteins and caveolins remains controversial. Here, we derived fibroblasts from Cav-1 null mouse embryos to study the behavior of GPI-linked proteins in the absence of caveolins. These cells lack morphological caveolae, do not express caveolin-1, and show a similar to95% down-regulation in caveolin-2 expression; these cells also do not express caveolin-3, a muscle-specific caveolin family member. As such, these caveolin-deficient cells represent an ideal tool to study the role of caveolins in GPI-linked protein sorting. We show that in Cav-1 null cells GPI-linked proteins are preferentially retained in an intracellular compartment that we identify as the Golgi complex. This intracellular pool of GPI-linked proteins is not degraded and remains associated with intracellular lipid rafts as judged by its Triton insolubility. In contrast, GPI-linked proteins are transported to the plasma membrane in wild-type cells, as expected. Furthermore, recombinant expression of caveolin-1 or caveolin-3, but not caveolin-2, in Cav-1 null cells complements this phenotype and restores the cell surface expression of GPI-linked proteins. This is perhaps surprising, as GPI-linked proteins are confined to the exoplasmic leaflet of the membrane, while caveolins are cytoplasmically oriented membrane proteins. As caveolin-1 normally undergoes palmitoylation on three cysteine residues (133, 143, and 156), we speculated that palmitoylation might mechanistically couple caveolin-1 to GPI-linked proteins. In support of this hypothesis, we show that palmitoylation of caveolin-1 on residues 143 and 156, but not residue 133, is required to restore cell surface expression of GPI-linked proteins in this complementation assay. We also show that another lipid raft-associated protein, c-Src, is retained intracellularly in Cav-1 null cells. Thus, Golgi-associated caveolins and caveola-like vesicles could represent part of the transport machinery that is necessary for efficiently moving lipid rafts and their associated proteins from the trans-Golgi to the plasma membrane. In further support of these findings, GPI-linked proteins were also retained intracellularly in tissue samples derived from Cav-1 null mice (i.e., lung endothelial and renal epithelial cells) and Cav-3 null mice (skeletal muscle fibers).