Schnell D J, Blobel G, Pain D
Laboratory of Cell Biology, Howard Hughes Medical Institute, Rockefeller University, New York, New York 10021.
J Cell Biol. 1990 Nov;111(5 Pt 1):1825-38. doi: 10.1083/jcb.111.5.1825.
A chloroplast import receptor from pea, previously identified by antiidiotypic antibodies was purified and its primary structure deduced from its cDNA sequence. The protein is a 36-kD integral membrane protein (p36) with eight potential transmembrane segments. Fab prepared from monospecific anti-p36 IgG inhibits the import of the ribulose-1,5-bisphosphate carboxylase small subunit precursor (pS) by interfering with pS binding at the chloroplast surface. Anti-p36 IgGs are able to immunoprecipitate a Triton X-100 soluble p36-pS complex, suggesting a direct interaction between p36 and pS. This immunoprecipitation was specific as it was abolished by a pS synthetic transit peptide, consistent with the transit sequence receptor function of p36. Immunoelectron microscopy localized p36 to regions of the outer chloroplast membrane that are in close contact with the inner chloroplast membrane. Comparison of the deduced sequence of pea p36 to that of other known proteins indicates a striking homology to a protein from spinach chloroplasts that was previously suggested to be the triose phosphate-3-phosphoglycerate-phosphate translocator (phosphate translocator) (Flügge, U. I., K. Fischer, A. Gross, W. Sebald, F. Lottspeich, and C. Eckerskorn. 1989. EMBO (Eur. Mol. Biol. Organ.) J. 8:39-46). However, incubation of Triton X-100 solubilized chloroplast envelope material with hydroxylapatite indicated that p36 was quantitatively absorbed, whereas previous reports have shown that phosphate translocator activity does not bind to hydroxylapatite (Flügge, U. I., and H. W. Heldt. 1981. Biochim. Biophys. Acta. 638:296-304. These data, in addition to the topology and import inhibition data presented in this report support the assignment of p36 as a receptor for chloroplast protein import, and argue against the assignment of the spinach homologue of this protein as the chloroplast phosphate translocator.
一种先前通过抗独特型抗体鉴定出的豌豆叶绿体输入受体被纯化，并根据其cDNA序列推导其一级结构。该蛋白是一种36kD的整合膜蛋白（p36），有八个潜在的跨膜区段。由单特异性抗p36 IgG制备的Fab通过干扰核酮糖-1,5-二磷酸羧化酶小亚基前体（pS）在叶绿体表面的结合来抑制其输入。抗p36 IgG能够免疫沉淀Triton X-100可溶性p36-pS复合物，表明p36与pS之间存在直接相互作用。这种免疫沉淀是特异性的，因为它被pS合成转运肽消除，这与p36的转运序列受体功能一致。免疫电子显微镜将p36定位到叶绿体外膜与叶绿体内膜紧密接触的区域。将豌豆p36的推导序列与其他已知蛋白的序列进行比较，发现它与菠菜叶绿体中的一种蛋白有显著同源性，该蛋白先前被认为是磷酸丙糖-3-磷酸甘油酸-磷酸转运体（磷酸转运体）（弗吕格，U.I.，K.费舍尔，A.格罗斯，W.泽巴尔德，F.洛茨皮希，和C.埃克斯科恩。1989.欧洲分子生物学组织杂志。8:39-46）。然而，用羟基磷灰石处理Triton X-100溶解的叶绿体被膜材料表明p36被定量吸收，而先前的报道表明磷酸转运体活性不与羟基磷灰石结合（弗吕格，U.I.，和H.W.黑尔德特。1981.生物化学与生物物理学报。638:296-304）。这些数据，除了本报告中呈现的拓扑结构和输入抑制数据外，支持将p36指定为叶绿体蛋白输入的受体，并反对将该蛋白在菠菜中的同源物指定为叶绿体磷酸转运体。
