Biermann B, Randall S K, Crowell D N
Department of Biology, Indiana University-Purdue University at Indianapolis 46202-5132, USA.
Plant Mol Biol. 1996 Aug;31(5):1021-8. doi: 10.1007/BF00040720.
To identify isoprenylated plant GTP-binding proteins, Arabidopsis thaliana and Nicotiana tabacum cDNA expression libraries were screened for cDNA-encoded proteins capable of binding [32P]GTP in vitro. ATGB2, an Arabidopsis homologue of the GTP-binding protein Rab2, was found to bind GTP in vitro and to be a substrate for a geranylgeranyl:protein transferase (GGTase) present in plant extracts. The carboxyl terminus of this protein contains a -GCCG sequence, which has not previously been shown to be recognized by any prenyl:protein transferase (PTase), but which most closely resembles that isoprenylated by the type II GGTase (-XXCC, -XCXC, or -CCXX). In vitro geranylgeranylation of an Arabidopsis Rab1 protein containing a carboxyl-terminal-CCGQ sequence confirmed the presence of a type II GGTase-like activity in plant extracts. Several other proteins were also identified by in vitro GTP binding, including Arabidopsis and tobacco homologues of Rab11, ARF (ADP-ribosylation factor) and Sar proteins, as well as a novel 22 kDa Arabidopsis protein (ATG81). This 22 kDa protein had consensus GTP-binding motifs and bound GTP with high specificity, but its structure was not closely related to that of any known GTP-binding protein (it most resembled proteins within the ARF/Sar and G protein alpha-subunit superfamilies).
为了鉴定异戊二烯化的植物GTP结合蛋白，对拟南芥和烟草的cDNA表达文库进行了筛选，以寻找能够在体外结合[32P]GTP的cDNA编码蛋白。发现拟南芥中GTP结合蛋白Rab2的同源物ATGB2在体外能结合GTP，并且是植物提取物中存在的香叶基香叶基：蛋白转移酶（GGTase）的底物。该蛋白的羧基末端包含一个-GCCG序列，此前尚未发现该序列能被任何异戊二烯基：蛋白转移酶（PTase）识别，但它与被II型GGTase异戊二烯化的序列（-XXCC、-XCXC或-CCXX）最为相似。对含有羧基末端-CCGQ序列的拟南芥Rab1蛋白进行体外香叶基香叶基化，证实了植物提取物中存在II型GGTase样活性。通过体外GTP结合还鉴定出了其他几种蛋白，包括Rab11、ARF（ADP核糖基化因子）和Sar蛋白的拟南芥和烟草同源物，以及一种新的22 kDa拟南芥蛋白（ATG81）。这种22 kDa蛋白具有一致的GTP结合基序，能高度特异性地结合GTP，但其结构与任何已知的GTP结合蛋白都没有密切关系（它与ARF/Sar和G蛋白α亚基超家族中的蛋白最为相似）。
