Lecordier L, Mercier C, Sibley L D, Cesbron-Delauw M F
Mécanismes Moléculaires de la Pathogénèse des Sporozoaires, Institut Pasteur de Lille, Institut de Biologie de Lille, 59019 Lille cedex, France.
Mol Biol Cell. 1999 Apr;10(4):1277-87. doi: 10.1091/mbc.10.4.1277.
The intracellular parasite Toxoplasma gondii resides within a specialized compartment, the parasitophorous vacuole (PV), that resists fusion with host cell endocytic and lysosomal compartments. The PV is extensively modified by secretion of parasite proteins, including the dense granule protein GRA5 that is specifically targeted to the delimiting membrane of the PV (PVM). We show here that GRA5 is present both in a soluble form and in hydrophobic aggregates. GRA5 is secreted as a soluble form into the PV after which it becomes stably associated with the PVM. Topological studies demonstrated that GRA5 was inserted into the PVM as a transmembrane protein with its N-terminal domain extending into the cytoplasm and its C terminus in the vacuole lumen. Deletion of 8 of the 18 hydrophobic amino acids of the single predicted transmembrane domain resulted in the failure of GRA5 to associate with the PVM; yet it remained correctly packaged in the dense granules and was secreted as a soluble protein into the PV. Collectively, these studies demonstrate that the secretory pathway in Toxoplasma is unusual in two regards; it allows soluble export of proteins containing typical transmembrane domains and provides a mechanism for their insertion into a host cell membrane after secretion from the parasite.
细胞内寄生虫刚地弓形虫寄生于一个特殊的区室，即寄生泡（PV）内，该寄生泡可抵抗与宿主细胞内吞和溶酶体区室的融合。寄生泡通过分泌寄生虫蛋白而被广泛修饰，包括致密颗粒蛋白GRA5，该蛋白特异性定位于寄生泡的界定膜（PVM）。我们在此表明，GRA5以可溶性形式和疏水聚集体形式存在。GRA5以可溶性形式分泌到寄生泡中，之后它与PVM稳定结合。拓扑学研究表明，GRA5作为跨膜蛋白插入PVM，其N端结构域延伸到细胞质中，C端位于泡腔内。预测的单个跨膜结构域的18个疏水氨基酸中的8个缺失导致GRA5无法与PVM结合；然而，它仍正确包装在致密颗粒中，并作为可溶性蛋白分泌到寄生泡中。总的来说，这些研究表明，弓形虫的分泌途径在两个方面是不同寻常的；它允许含有典型跨膜结构域的蛋白质进行可溶性输出，并提供了一种机制，使这些蛋白质在从寄生虫分泌后插入宿主细胞膜。
