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First published online 22 August 2006
doi: 10.1242/jcs.03148

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Arl1p is involved in transport of the GPI-anchored protein Gas1p from the late Golgi to the plasma membrane

Institute of Molecular Medicine, School of Medicine, National Taiwan University, and Department of Medical Research, National Taiwan University Hospital, Taipei 100, Taiwan

View larger version (56K): [in a new window]   Fig. 1. Arl1p is not directly involved in the recycling of GFP-Snc1p and the processing of vacuolar proteins. (A) GFP-Snc1p distribution in arl1-, gcs1- and ypt6-deleted yeast. The indicated deletion mutants of BY4741 (Invitrogen) were transformed with a plasmid expressing GFP-Snc1p. Transformants were grown to exponential phase and inspected under a Zeiss microscope. Maturation of vacuolar enzymes CPY (B) and ALP (C) in an arl1 mutant. Wild-type and arl1 mutant cells were grown, radiolabeled with [35S]methionine and [35S]cysteine at 37°C or 16°C and incubated for the indicated chase time (in minutes) before immunoprecipitation with anti-CPY (B) or anti-ALP (C) antibodies. Enzyme forms are indicated on the left. For CPY: p1, the core-glycosylated ER form; p2, outer-chain-glycosylated Golgi form; m, mature form resulting from proteolytic processing in the vacuole. For ALP: pro, proenzyme form of ALP; m, mature vacuolar form.

View larger version (50K): [in a new window]   Fig. 2. Arl1p is required for the surface targeting of GFP-GPI (Gas1p). (A) The location of GFP-GPI (Gas1p) and Gap1K9K16-GFP in arl1-, gcs1- and arl3-mutant cells. GFP-GPI (Gas1p) or Gap1K9K16-GFP was transformed into wild-type (WT), and arl1-, gcs1- and arl3-null cells and then induced in selection medium containing 2% galactose for 16 hours. GFP signals were observed as in Fig. 1A. (B) Distribution of GPI-anchored proteins. Crh1-GFP, Crh2-GFP, Cwp1-GFP or mRFP-Gas1 was transformed into wild-type and arl1-mutant cells. Transformants were grown overnight in selection medium and then transferred to fresh medium. After 3 hours of incubation, GFP signals were observed. (C) The intracellular accumulated mRFP-Gas1 in arl1 mutant cell colocalizes with GFP-Sft2. Plasmids expressing mRFP-Gas1 and GFP-Sft2 were co-transformed into wild-type and arl1-mutant cells. GFP and RFP signals were observed as described in B. Arrows indicate colocalization of mRFP-Gas1 and GFP-Sft2.

View larger version (60K): [in a new window]   Fig. 3. Arl1p is involved in transport of Gas1p from the late Golgi to the plasma membrane. (A) Congo Red hypersensitivity assay. Different mutant yeast were serially diluted tenfold as indicated and spotted on plates of YPD, Congo Red (100 µg/ml) and Congo Red (100 µg/ml) with 1.2 M sorbitol to analyze their sensitivity to cell-wall interference. (B) Steady-state distribution of Gas1p in arf1, arl1, gcs1, arl3 and wild-type cells. After mild digestion with lyticase, spheroplasts were treated with or without proteinase K to remove Gas1p that was localized at the plasma membrane. Residual Gas1p was detected with western blotting. For quantification, Gas1p signals were normalized to actin with the proteinase-K-untreated samples defined as 1. The fraction from the proteinase-K-treated samples indicated below each lane represents the intracellular and glycosylated Gas1p in each strain. Arrowheads indicate the ER unglycosylated form (105 kDa) of Gas1p. (C) Transport kinetics of Gas1p in arf1-, arl1-, gcs1- and arl3-mutant cells. Cells were pulse-labeled for 10 minutes with [35S]-Pro-mix and chased for 80 minutes. After removal of cell-surface proteins by treatment with proteinase K, Gas1p and actin were immunoprecipitated and analyzed by electrophoresis and autoradiography. Intracellular Gas1p was quantified as described in B. (D) Statistical analysis of the ratio of steady-state intracellular Gas1p in different mutant cells. Steady-state Gas1p distribution of three independent experiments was analyzed by plasma membrane proteinase shaving assay as described in B. (E) Statistical analysis of transport kinetics of intracellular Gas1p. Transport kinetics of Gas1p was traced by pulse-chase experiments as described in C. Error bars represent the mean ± s.e.m. of triplicate determinations.

View larger version (90K): [in a new window]   Fig. 4. Sys1p is involved in transport of Gas1p. (A) Congo Red hypersensitivity of the sys1 mutant. Different mutant cells (as indicated) were serially diluted and spotted on plates of YPD, Congo Red (100 µg/ml), and Congo Red (100 µg/ml) with sorbitol. A gas1 mutant was used as a control cell. (B) GFP-GPI (Gas1p) distribution is abnormal in arl1- and sys1-mutant cells. GFP-GPI (Gas1p) or Gap1K9K16-GFP were transformed into wild-type, arl1-, sys1- and arf3-null cells and then induced in selection medium containing 2% galactose for 16 hours. Living cells were imaged using a Zeiss microscope.

View larger version (63K): [in a new window]   Fig. 5. Subcellular distribution of endogenous Gas1p. (A) Differential centrifugation analysis. Wild-type or arl1-mutant cells were grown in YPAD medium, subjected to velocity sedimentation and then separated into P13 and S13 fractions as described in Materials and Methods. Proteins in samples of fractions were analyzed by immunoblotting. Gas1p, Pma1p (plasma membrane marker), Pgk1p (cytosol marker) and Kex2p (Golgi marker) were identified with specific antibodies. (B) Sucrose-density-gradient centrifugation analysis. S13 was layered on top of the sucrose gradient (10-30%), which was then subjected to centrifugation as described in Materials and Methods. Twelve fractions were collected from the top and samples were analyzed by western blotting using antibodies against Gas1p, Drs2p (late Golgi marker), Emp47p (early Golgi marker), Pgk1p (cytosol marker) and Pep12p (endosome marker). The lower panel displays the quantification of the western blot results.

View larger version (47K): [in a new window]   Fig. 6. Deletion of imh1, but not ypt6, affects transport of Gas1p to the plasma membrane. (A) Congo Red hypersensitivity of ypt6- and imh1-mutant yeast cells. Serial dilutions of different mutants, as indicated, were spotted on plates of YPD, Congo Red (100 µg/ml), and Congo Red (100 µg/ml) containing 1.2 M sorbitol to examine their hypersensitivity. (B) Intracellular Gas1p ratio in arl1-, imh1- and ypt6-mutant yeast cells. Intracellular Gas1p ratio of wild-type, arl1, imh1 and ypt6 mutants were analyzed with a plasma membrane proteinase shaving assay as described in Materials and Methods. The ratio of intracellular Gas1p was calculated as described in Fig. 3B. (C) Statistical analysis of steady-state intracellular Gas1p in different mutant cells. The intracellular Gas1p ratio of three independent experiments was determined as described in Fig. 3B. *P<0.05; error bars represent the mean ± s.e.m.

View larger version (94K): [in a new window]   Fig. 7. Arl1p regulates the integrity of the cell wall and localization of Gas1p in a GTP-dependent manner. (A) Congo Red hypersensitivity of an arl1 mutant overexpressing constitutively active Arl1p (Arl1Q72L), constitutively inactive Arl1p (Arl1T32N), and wild-type Arl1p. Different forms of Arl1p under the control of an ADH1 promoter were integrated into wild-type or arl1-mutant cells on the leu2 locus. Transformants were serially diluted and spotted on plates of YPAD, Congo Red (100 µg/ml), and Congo Red (100 µg/ml) containing 1.2 M sorbitol to examine their hypersensitivity. (B) The expression of constitutively inactive Arl1p (Arl1T32N) caused Congo Red hypersensitivity in wild-type cells. As described in A, different forms of Arl1p were introduced into wild-type cells and hypersensitivity to Congo Red was examined. (C) Effects of different forms of Arl1p in wild-type and arl1-mutant cells on localization of GFP-GPI (Gas1p). GFP-GPI was transformed into wild-type or arl1-mutant cells expressing different forms of Arl1p. After galactose induction, living cells were imaged using a Zeiss microscope.

View larger version (62K): [in a new window]   Fig. 8. Arl1Q72L localizes to the late Golgi in an arl3 mutant. (A) Mutant Arl1p localization in an arl3 mutant (upper panel). Different strains (as indicated) were stained with purified anti-Arl1p antibody and different ARL1 constructs were expressed in arl3-mutant yeast cells (lower panel); these ARL1 constructs were integrated into the leu2 locus in chromosomes. (B) Arl1Q72L colocalized with the late Golgi marker GFP-Sft2p in the arl3 mutant. Arl1Q72L and GFP-Sft2 were co-expressed in ARL3 and arl3 yeasts then stained with purified anti-Arl1p antibody and monoclonal anti-GFP antibody. Arrows indicate the colocalization of Arl1Q72L and GFP-Sft2.

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