Maize ROP7 GTPase contains a unique, CaaX box-independent plasma membrane targeting signal

Signals in the carboxy-terminal hypervariable region (HVR) of Rho and Ras G TPases target these proteins to specific membrane compartments, where they function in signal transduction. ROP6 and ROP7 are closely related maize Ro ps (a plant-specific Rho subgroup) that share HVR sequences divergent from other Rho HVRs. Both ROPs terminate in CAA, instead of the consensus C-term inal CaaX motif required for membrane association of all characterized Ras and Rho GTPases. The ROP6/7 HVR contains two additional cysteines, potentia l sites for post-translational modification that leads to membrane associat ion; 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 membran e (PM). Furthermore, the ROP7 HVR is sufficient to target GFP to the PM. Su rprisingly, the cysteine in the terminal CAA is not required for PM targeti ng of GFP-ROP7. In contrast, an internal HVR cysteine is essential for prop er targeting of the fusion, and the cysteine in the internal CaaX is requir ed for complete membrane association. Interestingly, this CaaX motif can al so direct PM association when placed at the fusion C-terminus by addition o f an internal stop codon. Fractionation experiments confirm that maize ROPs associate with membranes in maize seedlings. Our analysis suggests that th e ROP7 HVR directs PM localization by a mechanism independent of a C-termin al CaaX motif; this mechanism may have evolved through addition of 3' intro n/exon sequences to a rop progenitor.