Shnpsferllvetiyscooh Alg13C

FLAG-alg13-CA3 FLAG-ALG13 FLAG-alg13-CA3 FLAG-ALG13

FLAG-alg13-CA15D vector YPA + Galactose

FLAG-alg13-CA15 vector

FLAG-alg13-CA15 FLAG-ALG13 HA-ALG14

Alg13p

Lane: 1

IP: anti-HA; Blotting: anti-FLAG

Fig. 2a-c. The C-terminal a helix of yeast Alg13 is required for viability and for interaction with Alg14. a The C-terminal sequence of yeast Alg13 protein. The predicted a helix is shown in boldface and underlined. b Yeast strain (XGY154) with ALG13 under control of the glucose repressible GAL1 promoter (PGAL1) was transformed with plasmids containing FLAG-ALG13 (pXG211), FLAG-alg13-CA3 (pSA4), or FLAG-alg13-CA15 (pSA6), and streaked onto YPA plates supplemented with galactose (left panel) or glucose (right panel). Cells were incubated for 2 days at 30O C. c Whole-cell detergent extracts were prepared from a wild-type strain (W303a) that co-expresses HA-ALG14 (pXG202) and FLAG-alg13-CA15 (pSA6) or FLAG-ALG13 (pXG211). Samples were immunoprecipitated with anti-HA affinity matrix by Western blotting. Proteins or extracts were separated by 12% SDS-PAGE, immunoblotted with rabbit anti-FLAG antibodies, and detected by chemiluminescence as described in "Experimental Procedures"

because FLAG-alg13-CA15 failed to complement the growth of PGAL1-ALG13 strain XGY154 cells in glucose (Fig. 2b). These results suggested that the C-terminal a helix domain of Alg13 is essential for its function.

Our structural analysis suggested that the C-terminus of Alg13 was involved in its interaction with Alg14. To test whether this idea is correct, a co-immunoprecipitation assay was performed. Yeast strains were constructed that co-express N-terminally HA-tagged Alg14 with N-terminally FLAG-tagged Alg13-CA15p or wild-type Alg13p as a positive control (Fig. 2c). Detergent extracts were prepared from these strains and clarified by centrifugation at 100 000 g to remove any nonspecific protein aggregates (see "Experimental Procedures"). Alg14 proteins were immunoprecipitated with an anti-HA affinity matrix. The immuno-precipitates were separated by SDS-PAGE, and blotted with anti-FLAG rabbit antibodies to determine whether the truncated alg13-CA15p bound to Alg14. Unlike wild-type Alg13p, we found that HA-tagged Alg14 failed to bind FLAG-tagged Alg13-CA15p (Fig. 2c, lanes 1 and 2). Our failure to detect any bound Alg13-CA15p was not due to lowered intracellular levels of this protein, because Alg13 lacking its C-terminal 15 amino acids is as stable and as abundant as wildtype Alg13 protein in detergent extracts (data not shown). These results demonstrated that the C-terminal 15 amino acids of Alg13 are required for the interaction between Alg14 and Alg13 and suggest that the predicted a helix mediates this interaction.

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