Ivanchenko M, Vejlupkova Z, Quatrano R S, Fowler J E
Center for Gene Research and Biotechnology, Department of Botany, Oregon State University, Corvallis, OR 97331, USA.
Plant J. 2000 Oct;24(1):79-90. doi: 10.1046/j.1365-313x.2000.00855.x.
Signals in the carboxy-terminal hypervariable region (HVR) of Rho and Ras GTPases target these proteins to specific membrane compartments, where they function in signal transduction. ROP6 and ROP7 are closely related maize Rops (a plant-specific Rho subgroup) that share HVR sequences divergent from other Rho HVRs. Both ROPs terminate in CAA, instead of the consensus C-terminal CaaX motif required for membrane association of all characterized Ras and Rho GTPases. The ROP6/7 HVR contains two additional cysteines, potential sites for post-translational modification that leads to membrane association; one is in an internal CaaX motif, which would be at the C-terminus if the final intron in both genes were not removed. Transient expression of a GFP-ROP7 fusion revealed its near-total association with the plasma membrane (PM). Furthermore, the ROP7 HVR is sufficient to target GFP to the PM. Surprisingly, the cysteine in the terminal CAA is not required for PM targeting of GFP-ROP7. In contrast, an internal HVR cysteine is essential for proper targeting of the fusion, and the cysteine in the internal CaaX is required for complete membrane association. Interestingly, this CaaX motif can also direct PM association when placed at the fusion C-terminus by addition of an internal stop codon. Fractionation experiments confirm that maize ROPs associate with membranes in maize seedlings. Our analysis suggests that the ROP7 HVR directs PM localization by a mechanism independent of a C-terminal CaaX motif; this mechanism may have evolved through addition of 3' intron/exon sequences to a rop progenitor.
Rho和Ras GTP酶羧基末端高变区（HVR）中的信号将这些蛋白质靶向特定的膜区室，它们在那里参与信号转导。ROP6和ROP7是密切相关的玉米Rop蛋白（一种植物特有的Rho亚组），它们共享与其他Rho HVR不同的HVR序列。这两种Rop蛋白都以CAA结尾，而不是所有已鉴定的Ras和Rho GTP酶膜结合所需的共有C末端CaaX基序。ROP6/7 HVR包含另外两个半胱氨酸，这是导致膜结合的翻译后修饰的潜在位点；其中一个位于内部CaaX基序中，如果两个基因的最后一个内含子没有去除，该基序将位于C末端。GFP-ROP7融合蛋白的瞬时表达显示其几乎完全与质膜（PM）结合。此外，ROP7 HVR足以将GFP靶向到PM。令人惊讶的是，末端CAA中的半胱氨酸对于GFP-ROP7靶向PM不是必需的。相反，内部HVR半胱氨酸对于融合蛋白的正确靶向至关重要，内部CaaX中的半胱氨酸对于完全膜结合是必需的。有趣的是，当通过添加内部终止密码子将该CaaX基序置于融合蛋白C末端时，它也可以指导与PM的结合。分级分离实验证实玉米Rop蛋白与玉米幼苗中的膜结合。我们的分析表明，ROP7 HVR通过一种独立于C末端CaaX基序的机制指导PM定位；这种机制可能是通过向rop祖先添加3'内含子/外显子序列而进化而来的。
