TABLE 2.

Mutagenesis of potential sites for Smt3p interaction

Potential SUMO sites	Flanking sequence	Interaction with AD-SMT3
KAR9
K76	NKKRL GKDD ILLFM	+
K120	LEDEFS KDD QDSDK	+
K224, K227	TETTE PKVPK FSPA	+
K281, K282	QTILQ KKYE LIMKD	NDa
K301	SEFRE LKVE LIDKR	+
K307	SEFRE LKVE LIDKR	+
K301, K307	SEFRE LKVE LIDKR	+
K76, K120, K301, K307	NKKRL GKDD ILLFM	+
	LEDEFS KDD QDSDK
	SEFRE LKVE LIDKR
L304P	KVELIDKRWN	−
K529	SRGEN EKSP DSFIT	+
K594	ENTPI AKVF QTPPT	+
M293P	IMKDYRF M NSEFRE	−
BIM1
K25, K26	NLNYKKIEECGTG	+
K110	HWIRHKDESVYDP	+
K266, K267	LRFVKKVESILYA	+

The lysine residues (underlined) were mutated by site-directed mutagenesis in the KAR9-BD plasmid (pRM1493). In addition to the “standard” site identified by inspection (K301), additional potential SUMO attachment sites were identified by the SUMOplot Prediction software program (Abgent, http://www.abgent.com/tools/sumoplot). Leucine 304 lies immediately adjacent to a “standard” SUMO consensus site at K301. For the kar9 mutants, the following plasmids contain the indicated mutations: K76R (pRM4544), K120R (pRM6575), K224R K227R (pRM6576), K301R (pRM4472), K307R (pRM5415), K301RK307R (pRM6510), K76R K120R K301R K307R (pRM7831), L304P (pRM5421), K529R (pRM5424), K594R (pRM4698), and M293P (pRM6707). For the bim1 mutants, the plasmids are as follows: K25RK26R (pRM4699), K110R (pRM4700), and K266RK267R (pRM4701). Interaction with AD-SMT3 was tested as described in Figure 2, A and B. The boldface font represents residues of interest, such as potential sumoylation sites.

^aWe were unable to obtain the K281R K282R mutant, as either a single or double mutation. This is most likely due to the highly AT-rich region surrounding these residues.