Memon A R, Herrin D L, Thompson G A
Department of Botany, University of Texas, Austin 78713.
Biochim Biophys Acta. 1993 Oct 7;1179(1):11-22. doi: 10.1016/0167-4889(93)90070-6.
The primary aim of this study was to determine if small GTP-binding proteins play a role in the conspicuous and much-examined volume control process in Dunaliella salina. We confirmed the previous identification by Rodriguez et al. (Rodriguez Rosales, M.P., Herrin, D.L. and Thompson, G.A., Jr. (1992) Plant Physiol. 98, 446-451) of small GTP-binding proteins in the green alga Dunaliella salina and revealed the presence of at least five such proteins, having molecular masses of approx. 21, 28, 28.5, 29 and 30 kDa. These proteins were concentrated largely in the endoplasmic reticulum (ER) and in an intermediate density organelle fraction (GA) containing mainly Golgi vesicles, mitochondria and flagella. The chloroplast fraction and plasma membrane contained the 28-kDa GTP-binding protein exclusively, while the cytosol contained both the 28-kDa component and small amounts of a 21-kDa GTP-binding protein. Immunodetection analysis showed that the D. salina 28-kDa protein cross-reacted strongly with a polyclonal antibody raised against a Volvox carteri yptV1 type GTP-binding protein. This antibody was utilized for quantitative GTP-binding protein measurements as described below. Certain anti-GTP-binding protein antibodies derived from non-plant sources, namely, monoclonal antibodies raised against yeast and mouse ypt1 GTP-binding proteins, cross-reacted not only with the D. salina 28-kDa protein but also the 29-kDa component. The 30-kDa GTP-binding protein of D. salina did not bind the antibodies mentioned above but did cross-react with an anti-yeast ypt1 polyclonal antibody. None of the D. salina GTP-binding proteins reacted positively with polyclonal antibodies raised against SEC4, rab1 or rab6 proteins. When D. salina cells were subjected to hypoosmotic swelling by abruptly reducing the NaCl concentration of their medium from 1.7 M to 0.85 M, the increase in cell surface area was accompanied by a substantial translocation of the 28-kDa GTP-binding protein from the ER and GA fractions to the plasma membrane, chloroplast and cytosolic fractions, as determined by quantitative [32P]GTP binding and [125I]antibody binding on nitrocellulose blots. This translocation increased the content of the 28-kDa component in the plasma membrane, chloroplast and cytosol by 3-4-fold. No net movement of the 30-kDa GTP-binding protein from either the ER or GA fractions was observed following hypoosmotic shock.(ABSTRACT TRUNCATED AT 400 WORDS)
本研究的主要目的是确定小GTP结合蛋白是否在盐生杜氏藻中显著且备受研究的体积调控过程中发挥作用。我们证实了Rodriguez等人（Rodriguez Rosales, M.P., Herrin, D.L.和Thompson, G.A., Jr. (1992) Plant Physiol. 98, 446 - 451）之前对绿藻盐生杜氏藻中小GTP结合蛋白的鉴定，并揭示了至少五种此类蛋白的存在，其分子量约为21、28、28.5、29和30 kDa。这些蛋白主要集中在内质网（ER）和一个中等密度的细胞器组分（GA）中，该组分主要包含高尔基体小泡、线粒体和鞭毛。叶绿体组分和质膜仅含有28 kDa的GTP结合蛋白，而细胞质溶胶中既含有28 kDa的组分，也含有少量21 kDa的GTP结合蛋白。免疫检测分析表明，盐生杜氏藻的28 kDa蛋白与针对团藻yptV1型GTP结合蛋白产生的多克隆抗体发生强烈交叉反应。如下所述，该抗体用于定量GTP结合蛋白的测量。某些源自非植物来源的抗GTP结合蛋白抗体，即针对酵母和小鼠ypt1 GTP结合蛋白产生的单克隆抗体，不仅与盐生杜氏藻的28 kDa蛋白发生交叉反应，还与29 kDa的组分发生交叉反应。盐生杜氏藻的30 kDa GTP结合蛋白不与上述抗体结合，但与抗酵母ypt1多克隆抗体发生交叉反应。盐生杜氏藻的任何一种GTP结合蛋白都未与针对SEC4、rab1或rab6蛋白产生的多克隆抗体发生阳性反应。当通过将盐生杜氏藻细胞培养基中的NaCl浓度从1.7 M突然降至0.85 M使其遭受低渗肿胀时，细胞表面积的增加伴随着28 kDa GTP结合蛋白从ER和GA组分大量转运至质膜、叶绿体和细胞质溶胶组分，这是通过硝酸纤维素膜上的定量[32P]GTP结合和[125I]抗体结合确定的。这种转运使质膜、叶绿体和细胞质溶胶中28 kDa组分的含量增加了3 - 4倍。低渗休克后，未观察到30 kDa GTP结合蛋白从ER或GA组分有净移动。（摘要截于400字）
