Fig. 6. (A) Alignment of C-terminal amino acid sequences from various Glo3-type Arf GAPs. The ClustalW algorithm was used to create the sequence alignment. Identical amino acid positions for each Glo3-type ArfGAP are marked with asterisks (^*). Sc, S. cerevisiae; Hs, H. sapiens; Mm, M. musculus; Ce, C. elegans; Dm, D. melanogaster; At, A. thaliana. Note that one or two repeats of ISSxxxFG sequences exist in every Glo3-type ArfGAP. K386 and L411 of Glo3p are also conserved (shaded box). (B) Mutational analysis of the Glo3 motif. Various amino acid mutations were introduced into this motif of the chimeric protein 3HA-Glo3p. (C) NYY16-1 cells (arf1-16) were transformed with pYO324 (vector), pNY14-3HA-GLO3 (CEN 3HA-GLO3), pNY24-3HA-GLO3 (2 µ 3HA-GLO3), pNY24-glo3-m2HA (2 µ 3HA-glo3-11), pNY24-glo3-m1HA (2 µ 3HA-glo3-12), pNY24-glo3-m3HA (2 µ 3HA-glo3-13) and pNY24-glo3-m5HA (2 µ 3HA-glo3-14). Cells were grown, diluted and spotted on MCD (-Trp) plates, and incubated at the indicated temperatures for 2 days. Integrants of wild-type ARF1 (NYY0-1: WT) transformed with pYO324 were used as a control. (D) Immunoblotting of 3HA-Glo3p to examine expression levels. The transformants that were used in C were grown to an early log phase at 23°C, and incubated at 35°C for 1 hour. Total cell lysates (60 µg) were prepared and analyzed by immunoblotting using the anti-HA monoclonal and anti-Sec61p antibodies. Lane numbers correspond to the transformant numbers used in C.