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First published online 30 May 2006
doi: 10.1242/jcs.02997

Journal of Cell Science 119, 2604-2612 (2006)
Published by The Company of Biologists 2006
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The Arf1p GTPase-activating protein Glo3p executes its regulatory function through a conserved repeat motif at its C-terminus

N. Yahara1,*, K. Sato1,2 and A. Nakano1,3,{ddagger}

1 Molecular Membrane Biology Laboratory, RIKEN Discovery Research Institute, Hirosawa, Wako, Saitama 351-0198, Japan
2 PRESTO, Japan Science and Technology Agency, Hirosawa, Wako, Saitama 351-0198, Japan
3 Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan


Figure 1
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  		Fig. 1. Suppression activity of ArfGAPs, BET1, BOS1, OLE1, and GYP6 for arf1-16 and arf1-17 ts mutants. (A) arf1-16 (NYY16-1) and arf1-17 (NYY17-1) cells were transformed with pNY24-GLO3 (2 µ GLO3), pNY24-GCS1 (2 µ GCS1), pNY24-AGE1 (2 µ AGE1), pNY24-AGE2 (2 µ AGE2), and the transformants were grown at 23°C. Cells were diluted to a final concentration of 1.0 at OD 600, and 10 µl of 10-fold dilutions were spotted on MCD (minimal glucose casamino acid medium) (-Trp) plates. The plates were incubated at the indicated temperatures for 2 days. Integrants of wild-type ARF1 (WT, NYY0-1) and mutants transformed with pYO324 (vector) were used as controls. (B) arf1-16 and arf1-17 cells were transformed with pNY25-BET1 (2 µ BET1), pNY25-BOS1 (2 µ BOS1), pNY25-OLE1 (2 µ OLE1), and pNY25-GYP6 (2 µ GYP6), and the transformants were spotted on MVD (-Leu) plates and grown as described in A. Integrants of wild-type ARF1 (WT) and mutants transformed with pYO325 (vector) were used as controls.

 

Figure 2
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  		Fig. 2. Time course of [3H]GDP and [35S]GTP{gamma}S binding to myr-Arf1p and myr-arf1-17p. Purified Arf1p or arf1-17p (1.25 µM each) was mixed with 10 µM [3H]GDP (circle) or [35S]GTP{gamma}S (triangle) at 23°C (closed circles and triangles) and 30°C (open circles and triangles). At appropriate time intervals, aliquots of the reaction mixture were filtrated through a nitrocellulose membrane filter and then the radioactivity of the bound nucleotide was measured by scintillation counting.

 

Figure 3
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  		Fig. 3. Interactions of mutant arf1p with Arf GAPs in the DupLEX-ATM two-hybrid system. The interactions between N-terminally truncated (N17) Arf1p in the bait vector (pGlida) and Glo3p or Gcs1p in the prey vector (pJG4-5) were tested in the host strain EGY48. The Q71L mutation had been introduced in wild-type (ARF1), arf1-16 mutant (arf1-16) and arf1-17 mutant (arf1-17) genes. Transformants were plated on MVD (-Trp, -His), grown for 2 days, replicated on MVGal (minimal galactose medium) (-Trp, -His, -Leu), MVD (-Trp, -His), and MVD (-Trp, -His, -Leu) plates, and then further incubated at 30°C for 6 days. Empty prey vector (pJG4-5) was used as a negative control.

 

Figure 4
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  		Fig. 4. Time course of Arf GAP activity of Glo3p and Gcs1p. 0.2 µM recombinant Glo3p (circle) or Gcs1p (triangle) was incubated with 1 µM [{alpha}-32P]GTP-loaded WT-Arf1 (upper panel) or arf1-17 (lower panel) protein at 23°C (closed circle and triangle) and 35°C (open circle and triangle). Aliquots were withdrawn at the indicated times and subjected to the analysis of bound nucleotide by thin-layer chromatography.

 

Figure 5
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  		Fig. 5. (A) Sequence alignment of the N-terminal domains of Glo3p, Gcs1p and rat Arfgap1. The ClustalW algorithm was used to create the alignment. Asterisks indicates positions that have a single, fully conserved residue; colons and stops indicate that the stronger- and weaker-score groups is fully conserved, respectively. SAT motifs (C2C2H2) (Zhang et al., 1998Go) are underlined, and the ArfGAP domain of rat Arfgap1 (residues 1-136) (Cukierman et al., 1995Go; Goldberg, 1999Go) is shaded. (B) Construction of chimeric proteins of Glo3p and Gcs1p. (C) Suppression activities of Glo3p and Gcs1p chimeric proteins. arf1-16 cells were transformed with pNY24-GLO3 (Glo3p), pNY24-GCS1-A-GLO3 (Gcs1-A-Glo3p), pNY24-GCS1-N-GLO3 (Gcs1-N-Glo3p), pNY24-GLO3-N-GCS1 (Glo3-N-Gcs1p), pNY24-GLO3-A-GCS1 (Glo3-A-Gcs1p), and pNY24-GCS1 (Gcs1p), and the transformants were grown at 23°C. Cells were diluted, spotted on MCD (-Trp) plates, and incubated at the indicated temperatures for 2 days. Integrants of wild-type ARF1 (WT) and mutants transformed with pYO324 (vector) were used as controls.

 

Figure 6
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  		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.

 

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© The Company of Biologists Ltd 2006