Table II.

Site-directed Mutations in the GTP Binding Site of Srp102p

Allele		G-box location		Expression		Phenotype		Effect of mutation		Reference
Single amino acid changes in a single G-box
S49A		G-1		Yes		wild type		reduced GTP hydrolysis		Barbacid, 1987
K51I		G-1		Yes		ts		reduced nucleotide affinity		Pai et al., 1990
T52N		G-1		Yes		ts		increased affinity for GEF,
								reduced affinity for GTP		Peyroche et al., 1996
T66A		G-2		Yes		wild type		prevents GTP-dependent
								interaction with GAP		Sigal et al., 1986
G90L		G-3		Yes		ts		unknown
H91L		G-3		Yes		wild type		reduced GTPase activity		Jonak et al., 1994
N154I		G-4		Yes		ts		impaired nucleotide exchange		Sigal et al., 1986
E157Q		G-4		Yes		wild type		reduced affinity for GTP		Weijland et al., 1994
S220A		G-5		ND		wild type		bypass requirement for GEF		Camonis and Jacquet, 1988
Multiple amino acid changes in a single G-box
P46A,Q47A,N48A,ΔS49		G-1		Yes		wild type		unknown
K51A,T52A		G-1		Yes		wild type		unknown
N154A,E157A		G-4		Yes		wild type		unknown
Multiple amino acid changes in multiple G-boxes
H91L,N154I		G-3, G-4		Yes		ts		unknown
H91L,N154A,E157A		G-3, G-4		ND		ts		unknown
H91L,E157Q		G-3, G-4		Yes		ts		unknown
K51I,H91L		G-1, G-3		Yes		null		unknown
K51I,N154I		G-1, G-4		ND		null		unknown
K51A,T52A,H91L		G-1, G-3		Yes		null		unknown
K51A,T52A,E157Q		G-1, G-4		ND		null		unknown

Each site-directed mutation is listed along with its location in G-boxes 1–5 (nomenclature according to Bourne et al., 1991) and the observed growth phenotype when the mutant allele is present as the only copy of Srp102p is a haploid cell. Except for the mutants indicated by ND, wild-type expression levels of mutant proteins were confirmed by Western blot analysis. Most mutations were chosen on the basis of their predicted molecular phenotypes inferred from characterized defects in other GTPases as indicated.