Ras superfamily GTPase activation and inactivation occur by canonical nucleotide exchange and GTP hydrolysis mechanisms. Despite conservation of active-site residues, the Ras-related Rab GTPase activation pathway differs from Ras and between different Rabs. Analysis of DENND1-Rab35, Rabex-Rab5, TRAPP-Rab1 and DrrA-Rab1 suggests Rabs have the potential for activation by distinct GDP-release pathways. Conserved active-site residues in the Rab switch II region stabilising the nucleotide-free form differentiate these pathways. For DENND1-Rab35 and DrrA-Rab1 the Rab active-site glutamine, often mutated to create constitutively active forms, is involved in GEF mediated GDP-release. By contrast, in Rab5 the switch II aspartate is required for Rabex mediated GDP-release. Furthermore, Rab1 switch II glutamine mutants refractory to activation by DrrA can be activated by TRAPP, showing that a single Rab can be activated by more than one mechanistically distinct GDP-release pathway. These findings highlight plasticity in the activation mechanisms of closely related Rab GTPases.
The 70 or so members of the Rab subfamily of proteins perform a wide range of important tasks inside cells. A Rab protein is always bound to another molecule, which determines whether it is inactive or active. Binding to a molecule called GDP makes the Rab protein inactive, while binding to GTP makes it active. Proteins called guanine nucleotide exchange factors, or GEFs for short, activate the Rab protein by promoting the release of GDP and the binding of GTP. Other proteins—known as GAPs—lead to the inactivation of the Rab protein. Together these proteins form a molecular switch that can be turned on and off.
The Rab subfamily of proteins is part of the large Ras superfamily, and all members of this superfamily are activated and inactivated in a similar way, with the binding and unbinding of GDP and GTP taking place at a structure called the G-domain. The fact that the detailed structure of this domain (at the level of individual amino acids) has been conserved over evolution is often taken as an indication that its mechanism has also been conserved. Langemeyer et al. have now tested this assumption with four different types of GEFs—three from humans and one from the bacteria that cause Listeria—and found that the story is more complicated than expected.
The experiments showed that different amino acids in the active site of the Rab protein are involved when the GEFs mediate the release of the GDP during the activation process. For example, the amino acid glutamine is involved when the Listeria GEF and one of the human GEFs activate the protein, whereas a different amino acid—aspartate—is involved when one of the other human GEFs is responsible for the activation. Using this information, Langemeyer et al. create a human Rab protein that cannot be activated by the GEF from the bacteria that cause Listeria, but can still be activated by its normal human GEF.
By showing that different Rab proteins are activated by different mechanisms, and that a single Rab protein can be activated by more than one mechanism, the work of Langemeyer et al. clearly illustrates the on-going ability of evolution to surprise researchers.