Supplemental Figures

This article contains the following supporting material:

• Supplemental Figure 1 - Expression level of ARL1 and IMH1 constructs. (A) The expression level of Arl1-mRFP in wild-type cells. Arl1-mRFP expressed in wild -type cells was detected with an anti-Arl1p antibody and densitometry measurements of the bands indicated its expression level was ~3-fold higher than endogenous Arl1p. (B) The expression level of different ARL1 mutant constructs that were integrated into an arl1-deleted genome. The various mutant and wild-type Arl1p were detected with anti-Arl1p antibodies. The expression level of these mutant Arl1p proteins was approximately 3-5 fold higher than endogenous Arl1p. (C) The expression level of GFP-Imh1p in wild-type yeast. GFP-IMH1pRS314 was transformed into wild-type yeast and cells were induction in 2% galactose and 0.1 % glucose medium for 12 hours, GFP-Imh1p was detected with an anti-Imh1p antibody.
• Supplemental Figure 2 - Gcs1p does not interact with Arf1d17N, Arl3d17N and Arl3d17NQ78L. GCS1-pJG4-5 and different ARF1/ARL3-pEG202 constructs were co-transformed into YEM1α and spotted on X-gal plate as described in Figure 1. Lower panels were the expression of these fusion proteins. LexA-fused ARF/ARLs were detected by Anti-LexA antibody, HA-taged Gcs1p by anti-HA, and Imh1p was used as control.
• Supplemental Figure 3 - Arl1Q72L is adjacent to the vacuole. Arl1Q72L from integrated plasmid YIplac128 under the control of the ADH promoter was transformed into arl1 (YPH250dl1). Logarithmically growing cells were fixed and spheroplasts were prepared for reaction with affinity-purified anti-Arl1p antibody and monoclonal anti-v-ATPase antibody. Indirect immunofluorescence staining was done as described in Materials and Methods. Observations were replicated in at least three experiments.
• Supplemental Figure 4 - More Arl1-mRFP is present in trans-Golgi of arl1gcs1 mutant than arl1 cell. (A) arl1 or arl1gcs1 mutant cells expressing GFP-Sft2 from pRS314 and Arl1-mRFP from pRS315 vectors were observed in living cells. (B) Golgi association ratio of Arl1mRFP inarl1or arl1gcs1 mutant cells. The percentage of Golgi-associated protein was quantified with AxioVision Rel. 4.2 software. Observations were replicated in at least three experiments.
• Supplemental Figure 5 - The effect of over-expression of different GAPs on the localization of Arl1p or Arf1p. (A) Gcs1-GFP, but not Gcs1-zn-GFP over-expression causes Arl1-mRFP to diffuse into cytosol. Gcs1-GFP or Gcs1-zn-GFP on pVT101U and Arl1-mRFP (pRS315) were co-transformed into arl1gcs1-null cells and living cells were observed and photographed. The over-expression of Gcs1-GFP did not apparently disturb the Golgi distribution of Arf1-mRFP. Gcs1-GFP (pVT101U) and Arf1-mRFP (pRS315) were co-transformed into gcs1-deleted yeast. Mid-log yeast were observed with microscope. (B) Glo3-myc or Age2-myc over-expression did not affect Arl1p location. Glo3-myc or Age2-myc on pVT101U was transformed into wild-type yeast then stained as described.