The small GTPases of the Ras superfamily are involved in regulating many intracellular processes, including cell growth and division, cell morphology and movement, vesicular transport, and nuclear events (4
). These proteins, which act as molecular switches to control various functions in the cell, are in the active, or “on,” state when bound to GTP and the inactive, or “off,” state when bound to GDP. The immediate control of these GTPase-mediated events resides in the proteins which regulate their GTP- or GDP-binding status. Two classes of regulatory proteins have been identified: the guanine nucleotide exchange factors (GEFs), whose physiological function is to convert GTPases from a GDP-bound state to a GTP-bound state, and the GTPase-activating proteins (GAPs), which turn off the GTPases by activating an intrinsic GTPase activity (3
). The GEFs stimulate guanine nucleotide release to yield a GEF–apo-GTPase reaction intermediate and, in part because the GTP concentration in cells is higher than that of GDP, the formation of active GTP-bound GTPase is favored (61
Most of our understanding of the physical interaction of these regulatory molecules with the small GTPases is based on studies of the Ras protein (3
). For example, it is known that Ras GAPs bind to the effector loop of Ras (3
). The Ras effector loop, comprising residues 30 to 45, also interacts with the known downstream targets of Ras (42
Numerous groups have contributed to the effort to identify Ras residues which are involved in interactions with GEFs. Residues 62 to 75 in the switch II region of H-ras were found to be involved, as were residues 103 and 105 in the alpha-helix 3–loop 7 (α3-L7) region (16
). The effector loop (switch I region) of Ras was also implicated in direct interactions with GEFs (5
). The switch I, switch II, and α3-L7 regions of H-ras are found adjacent to each other on the surface of the molecule, as would be expected for a surface domain involved in GEF binding (see Fig. ) (36
). The recently described crystal structure of H-ras complexed with Sos demonstrates that each of these three regions is indeed at the interface of the Ras-Sos complex (5
FIG. 7 Diagram showing the structure of H-ras bound to GDP. The effector loop (residues 35 to 42) (magenta), switch II region (residues 62 to 76) (cyan blue), and α3-L7 region (residues 101 to 109) (green) of Ras are on the surface of the molecule and (more ...)
Ras GEFs exhibit a modest preference for binding GDP-bound forms of Ras, whereas Ras GAPs preferentially bind GTP-bound forms (28
). Thus, the GEFs and GAPs which affect the nucleotide-binding status of Ras preferentially bind their respective substrates rather than their products. The high affinities for substrates likely reflect structural differences between the two nucleotide-bound forms of Ras. Significantly, the switch I and switch II regions of H-ras, known to have altered structures when bound to either GDP or GTP, fall within the regions implicated in interactions with GEFs and GAPs (66
Recently, the crystal structure of the Sec7 domain of human Arno, a GEF for the Arf GTPase, and an analysis of the interaction sites of these two proteins have been reported (48
). The analysis revealed that Arf interacts with its exchange factor in a manner reminiscent of the Ras interaction with its GEFs. Arf appears to use three noncontiguous segments of its polypeptide to interact with Sec7. Importantly, these three regions of the Arf protein are analogous to those used by Ras to interact with its GEFs. The switch I region (effector loop) and switch II region of Arf and Ras interact with their GEFs (5
). Also, Ras residues 103 to 105 in the α3-L7 region and the corresponding residues of Arf (residues 113 to 115) appear to bind GEFs (5
). While the GEF-binding sequences of Arf and Ras are at analogous positions in the GTPases, GEF-binding sequences of Ras do not show homology with the Arf sequences. The finding that these two distantly related GTPases use analogous regions to interact with their GEFs raises several questions relating to other subclasses of GTPases. For example, do the Rho and Rab/YPT1 families of GTPases interact with their GEFs by using domains analogous to those used by Ras and Arf? Do the different families of GEF use a similar mechanism for catalyzing guanine nucleotide exchange on small GTPases?
We undertook the present study to ask whether other small GTPases use the regions corresponding to the GEF-binding domain of H-ras to interact with their cognate GEFs. For this study, we chose the yeast YPT1 protein, which is a member of the Rab family of small GTPases (22
). This family of proteins is involved in regulating vesicular transport (54
). Previously we used a yeast genetic screen to identify Ras residues which were involved in binding to Ras GEFs (49
). This screen uses both a dominant interfering mutant and a constitutively active mutant of Ras. Here we created analogous YPT1 mutants and demonstrated that they could be used in a similar genetic screen. We demonstrated that the mechanism of dominant interference of YPT1 mutant N22 (YPT1-N22) is sequestration of an endogenous essential GEF for YPT1 such that a lethal phenotype occurs because endogenous YPT1 cannot be activated. Using both site-directed and random mutagenesis procedures, we identified a series of intragenic suppressors of YPT1-N22, among which we predicted would be mutants which fail to sequester essential GEFs for YPT1 due to the loss of a complete GEF-binding domain.
Among the intragenic suppressor mutations, we identified 10 residues, at positions 42, 43, 49, 69, 71, 73, 75, 107, 109, and 115, which were involved in in vitro binding to DSS4, a GEF which can stimulate nucleotide exchange on YPT1 in vitro (10
). The positions of these residues correspond to the switch I, switch II, and α3-L7 regions of Ras, the same regions found to be important for Ras interaction with GEFs.
Our findings suggest that the interaction of Ras with its specific GEFs may prove to be a useful model for analyzing the structural basis underlying the interaction of other small GTPases with their cognate GEFs. Further, our findings, together with an analysis of the interactions of Ras and Arf GTPases with their GEFs, indicate that small GTPases of the Ras superfamily use similar regions for interactions with GEFs, suggesting a similar catalytic mechanism of guanine nucleotide exchange for all small GTPases.