Analysis of single deletion mutants of the ChAPs together with mutants in CHS5 and ARF1. For the drop assays, haploid strains were grown overnight to logarithmic phase in rich medium. Serial dilutions (1:10) were dropped onto plates and incubated for 2 days at 30°C, unless indicated otherwise. (A) Growth at different temperatures. (B) Growth on YMP+ plates, which contain an elevated level of ammonium sulfate. (C) Analysis of the budding pattern. Diploid yeast strains were grown to logarithmic phase and stained with calcofluor. (D) Analysis of the budding pattern of mutants in CHS5 and CHS6. Diploid yeast strains were grown to logarithmic phase in YPD+0.7 M sorbitol. After fixation, cells were stained with FITC–concanavalin A. (E) Growth on minimal medium plates buffered at pH 5.8 and 7.5. (F) Scheme depicting the relationship of the members of the ChAP family. The percentage of protein sequence identity is given.

Chs5p and members of the ChAPs are involved in transport of chitin synthase Chs3p to the plasma membrane. (A) Haploid strains were grown to logarithmic phase in rich medium. Serial dilutions (1:10) were dropped onto plates containing the toxic dye calcofluor and incubated for 2 days. (B) Haploid yeast cells bearing a chromosomal CHS3-GFP were grown to logarithmic phase. Arrowheads point to the signal at the bud neck and the incipient bud site. Pictures were taken from freshly mounted cells and two images per strain are shown.

The ChAPs are at least partially localized to the TGN. The ChAPs were chromosomally tagged with 9myc and Sec7p with GFP. Strains were grown to logarithmic phase, fixed and stained with α-myc and α-GFP antibodies. α-Myc and α-GFP were detected with Cy3 and FITC, respectively.

The ChAPs interact with Arf1p and Chs5p. Co-immunoprecipitation experiments were performed using strains in which one ChAP was individually chromosomally tagged with 9myc in either WT, in Δchs5 or in a strain in which the three remaining ChAPs were deleted. Furthermore, the experiment was also performed with lysates from ChAPΔc-9myc strain. The lysates were treated with either affinity-purified α-Arf1p (A) or α-Chs5p serum (B) and Protein A–Sepharose. The precipitate was analyzed with α-myc. In all, 1.7% of the lysates was loaded in lanes 1–4. The lysates in (A) and (B) are from the same experiment. The lysates and corresponding precipitates were from the same immunoblot.

Chs3p interacts with the ChAPs. (A) Crosslinking experiments were performed using either WT or Δchs5 strains carrying Chs6p-9myc. The lysates were treated with either DMSO or DSP before Chs3p immunoprecipitation. The precipitate was analyzed by immunoblot with α-myc. In all, 8% of the lysates was loaded in lanes 1 and 2. (B) Crosslinking experiments were performed using strains in which the ChAPs were chromosomally tagged with 9myc as in (A). The lysates and corresponding precipitates were from the same immunoblot, and image processing was identical.

Chs5p binds to activated Arf1p. (A) Differential Arf1p affinity chromatography to evaluate binding to Arf1p. Yeast cytosol was incubated with either Arf1p-Q71L (Arf1p-GTP) or Arf1p-T31N (Arf1p-GDP) column material. After washing, spontaneous nucleotide exchange was elicited resulting in a conformational change on Arf1p and the release of conformation-specific bound proteins. The eluates (E1–E3) from an affinity chromatography experiment were analyzed by immunoblot. FT is the flow-through of unbound protein. Beads without Arf1p were mock-treated and served as negative control. The Arf1p-GEF Gea2p is enriched in the Arf1p-GDP column, whereas both the coatomer complex (three subunits are shown) and the Arf1p-GAP Glo3p bind predominately to Arf1p-GTP. (B) Chs5p binds to activated Arf1p. Eluates (E1–E4) from the Arf1p affinity chromatography were analyzed for the presence of Chs5p by immunoblot. (C) The ChAPs interact with Arf1p. Co-immunoprecipitation experiments were performed using strains in which the ChAPs were chromosomally tagged with 9myc. The lysates were treated with affinity-purified α-Arf1p-IgGs or α-Arf1p and α-Sar1p sera and Protein A–Sepharose. The precipitate was analyzed by immunoblot with antibodies directed against the myc epitope, Sar1p and Arf1p. Lanes 1 and 4 and lanes 7 and 10 represent 1.3 and 1.7% of the lysate, respectively. The lysates and corresponding precipitates were from the same immunoblot. Image processing was identical for lysate and corresponding precipitates. (D) Bch2p interacts directly with Arf1p. Liposomes were incubated with Bch2p in the presence or absence of Arf1p-GTP. The liposomes were floated through a sucrose cushion and the top layer was analyzed by immunoblot against Arf1p and Bch2p. The flotation of coatomer served as control and Cop1p was detected by immunoblot. In all, 10% of the input of Arf1p, coatomer and Bch2p were loaded for comparison.

The ChAPs interact with each other. A quadruply chromosomally tagged strain along with control strains were subjected to immunoprecipitation. The precipitates were analyzed by SDS–PAGE and immunoblot with antibodies directed against all four different epitope tags. Precipitations with α-AU5 and with α-myc-IgGs are shown. In all, 1.7% of the lysates were loaded in lanes 1, 2 and 5–7. The lysates and corresponding precipitates were from the same immunoblots. Image processing was identical for lysates and corresponding precipitates.

The C-terminus of the ChAPs is essential for their function. (A) Sequence alignment of the C-terminus of the ChAPs. The last 13 amino acids of Chs6p were deleted and replaced by 9myc. The C-termini of the other three family members were deleted and replaced by epitope tags according to this alignment. A dashed line indicates the site of deletion. (B) Growth of CHS6Δc-9myc as compared to WT and Δchs6 at different temperatures and on calcofluor plates. (C) Budding pattern of a homozygous diploid BUD7Δc-9myc strain. Diploid yeast strains were grown to logarithmic phase and stained with calcofluor. (D) Growth of BCH1Δc-3HA as compared to WT and Δbch1 at different temperatures and on YMP+ plates.

Chs5p is essential for the TGN localization of the ChAPs. (A) Chs5p-6HA staining in WT cells and in cells in which the whole ChAP family is deleted (ΔΔΔΔ). (B) Staining of ChAP-9myc in Δchs5 strains or strains in which the other three ChAPs were deleted (ΔΔΔ) or the ChAPΔc-9myc in a WT background. The same exposure time as in Figure 5 was used for each protein within one row.