Sotgia Federica, Razani Babak, Bonuccelli Gloria, Schubert William, Battista Michela, Lee Hyangkyu, Capozza Franco, Schubert Ann Lane, Minetti Carlo, Buckley J Thomas, Lisanti Michael P
Department of Molecular Pharmacology, The Albert Einstein Cancer Center, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Mol Cell Biol. 2002 Jun;22(11):3905-26. doi: 10.1128/MCB.22.11.3905-3926.2002.
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 approximately 95% 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).
糖基磷脂酰肌醇（GPI）连接蛋白与小窝蛋白之间的关系仍存在争议。在此，我们从Cav-1基因敲除小鼠胚胎中获取成纤维细胞，以研究在缺乏小窝蛋白的情况下GPI连接蛋白的行为。这些细胞缺乏形态学上的小窝，不表达小窝蛋白-1，且小窝蛋白-2的表达下调约95%；这些细胞也不表达小窝蛋白-3，后者是一种肌肉特异性的小窝蛋白家族成员。因此，这些缺乏小窝蛋白的细胞是研究小窝蛋白在GPI连接蛋白分选过程中作用的理想工具。我们发现，在Cav-1基因敲除细胞中，GPI连接蛋白优先保留在一个我们确定为高尔基体复合体的细胞内区室中。这个细胞内的GPI连接蛋白池不会被降解，并且根据其对 Triton 的不溶性判断，它仍然与细胞内脂质筏相关。相比之下，正如预期的那样，GPI连接蛋白在野生型细胞中被转运到质膜。此外，在Cav-1基因敲除细胞中重组表达小窝蛋白-1或小窝蛋白-3，但不包括小窝蛋白-2，可以补充这种表型并恢复GPI连接蛋白的细胞表面表达。这可能令人惊讶，因为GPI连接蛋白局限于膜的外质小叶，而小窝蛋白是面向细胞质的膜蛋白。由于小窝蛋白-1通常在三个半胱氨酸残基（133、143和156）上进行棕榈酰化，我们推测棕榈酰化可能在机制上将小窝蛋白-1与GPI连接蛋白联系起来。为支持这一假设，我们表明在这种互补实验中，小窝蛋白-1在残基143和156上的棕榈酰化，而不是残基133上的棕榈酰化，是恢复GPI连接蛋白细胞表面表达所必需的。我们还表明，另一种与脂质筏相关的蛋白c-Src在Cav-1基因敲除细胞中保留在细胞内。因此，与高尔基体相关的小窝蛋白和类小窝囊泡可能代表了将脂质筏及其相关蛋白从反式高尔基体有效转运到质膜所必需的运输机制的一部分。为进一步支持这些发现，GPI连接蛋白在源自Cav-1基因敲除小鼠（即肺内皮细胞和肾上皮细胞）和Cav-3基因敲除小鼠（骨骼肌纤维）的组织样本中也保留在细胞内。
