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First published online May 4, 2004
doi: 10.1242/10.1242/jcs.01050

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Yeast ARL1 encodes a regulator of K⁺ influx

Department of Biology, 406 Reiss Science Center, Box 571229, Georgetown University, Washington, DC 20057, USA

View larger version (86K): [in a new window]   Fig. 4. Mutants deleted for genes obtained in the high-copy-number suppressor screen are variously sensitive to hygromycin B. Selected homozygous diploid deletion strains (from the Deletion Collection) were grown overnight in YPAD liquid medium to saturation. Serial tenfold dilutions in water were performed in a multiwell plate and then spotted onto solid medium with a replicator tool. Strains were tested on YPAD medium with or without 0.1 mg ml–1 hygromycin B and with or without 100 mM KCl. Cells grown in YPAD + KCl grew as well as cells on YPAD alone and are therefore not shown. Cells were grown for 4 days at 30°C then photographed.

View larger version (39K): [in a new window]   Fig. 1. Strains lacking ARL1 are sensitive to hygromycin B and TMA. The parental strains PSY316 (ARL1; left) and MA03 (arl1; right), were struck onto YPAD medium (Adams et al., 1997; Sherman et al., 1974) containing no additions (left), 0.1 mg ml–1 hygromycin B (middle) or 0.25 M TMA (right), without other additions (top) or with the addition of 100 mM KCl (bottom). The plates were incubated for 3 days at 30°C and photographed.

View larger version (38K): [in a new window]   Fig. 2. Alleles of ARL1 and their ability to complement the hygromycin-B-sensitive phenotype of arl1 strains. The arl1 strain MA03 was transformed with the constructs listed, then grown overnight in minimal medium lacking uracil. The overnight cultures were subjected to tenfold serial dilutions in water and then spotted onto YPAD plates without (left) or with (right) hygromycin B (0.1 mg ml–1) using a replicator tool. The plates were incubated at 30°C for 3 days and photographed.

View larger version (9K): [in a new window]   Fig. 3. Strains lacking ARL1 accumulate [14C]-MA. PSY316 (ARL1; squares) and MA03 (arl1; circles) were incubated with [14C]-MA (2.5 µCi ml–1, 2 mM final concentration) for up to 60 minutes. Cells were collected on nitrocellulose filters and washed three times with ice-cold 20 mM MgCl2. Filters were counted by scintillation spectroscopy to determine the amount of internalized radioactivity. Data were collected in triplicate and are shown ±s.d. This experiment was repeated four times with similar results.

View larger version (19K): [in a new window]   Fig. 5. Strains lacking ARL1 have a 86Rb+ uptake defect. (A) PSY316 (ARL1; squares) and MA03 (arl1; circles) were treated then incubated with 86RbCl for up to 45 minutes as described in Materials and Methods. Uptake by each strain was measured in triplicate and data shown is the average±s.d. This experiment was repeated three times with similar results. (B) Selected strains from the Deletion Collection were treated then incubated with 86RbCl for 0 minutes and 30 minutes as described in Materials and Methods. Uptake by each strain was measured in triplicate and data shown in the average difference ± s.d. between the 0 and 30 minute time points. This experiment was repeated twice with similar results.

View larger version (15K): [in a new window]   Fig. 6. Strains lacking ARL1 do not have an apparent H+ efflux defect. PSY316 (ARL1; squares) and MA03 (arl1; circles) were treated as described in Materials and Methods. Measurements made in the presence of 25 mM KCl are open symbols and without KCl are closed symbols. Proton efflux was determined by measuring the change in pH after initiating the reaction with glucose. This experiment was repeated three times with similar results.

View larger version (48K): [in a new window]   Fig. 7. Strains lacking ARL1 do not mislocalize Trk1-Myc. AM300 (TRK1-Myc ARL1) and AM310 (TRK1-Myc arl1) were grown in SD medium with supplements to mid-log phase and then harvested by centrifugation and prepared for subcellular fractionation as described in Materials and Methods. Equal numbers of cell equivalents (as measured by OD600), after spheroplasting and lysis, were fractionated by differential centrifugation. After addition of SDS-PAGE sample buffer followed by heating to 42°C for 5 minutes, samples were loaded onto a 10% polyacrylamide gel. After electrophoresis, the proteins were electroblotted to nitrocellulose, then used for western-blot analysis with anti-Pgk1p antibody (top panel; a cytosol marker), anti-Pma1p antibody (second panel; a plasma membrane marker), anti-Vph1p antibody (third panel; vacuole marker) or anti-Myc antibody to detect Trk1p-Myc (bottom panel).