The Functional Specialization of Exomer as a Cargo Adaptor During the Evolution of Fungi

Abstract

Yeast exomer is a heterotetrameric complex that is assembled at the trans-Golgi network, which is required for the delivery of a distinct set of proteins to the plasma membrane using ChAPs (Chs5-Arf1 binding proteins) Chs6 and Bch2 as dedicated cargo adaptors. However, our results show a significant functional divergence between them, suggesting an evolutionary specialization among the ChAPs. Moreover, the characterization of exomer mutants in several fungi indicates that exomer's function as a cargo adaptor is a late evolutionary acquisition associated with several gene duplications of the fungal ChAPs ancestor. Initial gene duplication led to the formation of the two ChAPs families, Chs6 and Bch1, in the Saccaromycotina group, which have remained functionally redundant based on the characterization of Kluyveromyces lactis mutants. The whole-genome duplication that occurred within the Saccharomyces genus facilitated a further divergence, which allowed Chs6/Bch2 and Bch1/Bud7 pairs to become specialized for specific cellular functions. We also show that the behavior of S. cerevisiae Chs3 as an exomer cargo is associated with the presence of specific cytosolic domains in this protein, which favor its interaction with exomer and AP-1 complexes. However, these domains are not conserved in the Chs3 proteins of other fungi, suggesting that they arose late in the evolution of fungi associated with the specialization of ChAPs as cargo adaptors.

Keywords: evolution; exomer; intracellular traffic; yeast.

Figure 1

Phenotypes of BCH2 overexpression. (A) Calcofluor white (CW) resistance promoted by multicopy plasmids pRCW3 and pJV30, both containing BCH2. (B) CW vital staining of the indicated strains. Note the reduction of fluorescence after BCH2 overexpression. (C) Localization of Chs3-GFP in chs3∆ strains transformed with the indicated plasmids. Numbers indicate the percentage of cells showing localization at the neck (n > 100). (D) CW resistance of the indicated mutants transformed with control or pJV30 plasmids. Note the sensitization to CW in the aps1∆ mutant after BCH2 overexpression. These experiments were always performed in strain CRM1590 (chs3Δ::natMx4) transformed with plasmids pRS315 or pRS315-Chs3-GFP as indicated. (E) Localization of Chs5-mCh and Chs6-mCh after BCH2 overexpression (pJV30). Chs5-mCh and Chs6-mCh are tagged on the chromosome. (F) CW resistance after overexpression of the different ChAPs. Experiment was carried out in wild-type (WT) cells in which the pGAL promoter was inserted at the chromosome replacing the endogenous promoter of each ChAP. Overexpression of the different ChAPs was achieved by growth in galactose-supplemented media. Note that cells grown in glucose should behave similarly to null mutants of the corresponding genes. Also see Figure S2 in File S1 for a more complete set of experiments.

Figure 2

Phylogeny of ChAPs (Chs5-Arf1 binding proteins) along fungi. (A) Phylogenetic tree of the Saccharomycotina clade. Major evolutionary lineages are indicated, including the proposed EB groups. Tree images were obtained from MycoCosm portal (Grigoriev et al. 2014). The number of ChAPs members identified in each group is indicated on the right. Note the presence of a single ChAP in all early branched genera, which is similar to other major groups of fungi (see also Figure S4 in File S1). (B) A phylogenetic analysis of the ChAPs family. Individual proteins were identified by BLAST analysis and a multiple alignment with CLUSTALW was later performed. Analysis is represented as a rooted phylogenetic tree (UPGMA) with branch lengths. Only the genes within the genus Saccharomyces have been named, and the letters A and B have been used to indicate the homologous closest to ScBch1 and ScChs6, respectively. See text for a more detailed description of the tree. BLAST, basic local alignment search tool; CTG, species that translate CTG codon as serine; EB, early branched; WGD, whole-genome duplication.

Figure 3

Characterization of exomer mutants in different fungi. (A) Sensitivity of the chs5∆ mutants of different fungi to calcofluor white (CW) and caspofungin, as compared to the corresponding wild-type (WT) and chs3∆ mutants used as controls. chs3∆ mutants were resistant to CW and sensitive to caspofungin in all organisms, but only the S. cerevisiae chs5∆ exomer mutant reproduced this phenotype. (B) CW staining of fixed cells on the indicated strains and organisms. Note the absence of chitin rings in all the chs3∆ mutants, which did not occur in the chs5∆ mutant, except in S. cerevisiae. U. maydis chs5∆ mutant showed normal CW vital staining. (C) Extended characterization of phenotypes of the different mutants. Cells were grown to early logarithmic phase, serial diluted, and plated on the indicated media. Growth was score after 2–3 days of growth at 28°.

Figure 4

Phenotypic characterization of exomer mutants in S. cerevisiae and K. lactis. Growth of the indicated S. cerevisiae (A) and K. lactis (B) mutants on YEPD gradient plates containing increasing concentrations of different compounds. Logarithmic cultures were diluted OD₆₀₀ 0.1 and spotted at identical concentrations along the gradient plate. (C) Growth of the indicated S. cerevisiae strains on complex media supplemented with NH₄Cl. (D) Growth of the indicated K. lactis mutants on YES media supplemented with the indicated nitrogen sources. Panel on the right represents the plate supplemented with Lys, where the results with either amino acid were identical. (C and D) show early logarithmic phase cultures grown on YEPD that were serial diluted and spotted onto the different plates. Note the specific sensitivity of S. cerevisiae exomer mutants to high concentrations of NH₄Cl, which is not observed in the corresponding mutant in K. lactis. The individual ChAPs mutants of S. cerevisiae did not showed sensitivity toward any of the compounds used in this figure (see Figure S2B in File S1), therefore only the double ChAPs mutants were tested. K. lactis contains only two ChAPs, therefore the results for the individual mutants are presented. Note that in S. cerevisiae always one of the double mutants behaved exactly as the null exomer mutant chs5∆, while in K. lactis the individual mutants lacked phenotype and only the absence of the two ChAPs showed a phenotype equivalent to the null chs5∆ mutant. YEPD, 1% Bacto yeast extract, 2% peptone, and 2% glucose; YES, yeast extract, glucose, and supplements; WT, wild-type.

Figure 5

Characterization of chitin synthesis in C. albicans. (A) CW vital staining of C. albicans yeast cells strains as indicated (upper panels). Staining was performed for 60 min. For hyphal visualization (lower panels), filamentation was induced for 2 hr and staining was performed on fixed cells as described in the Materials and Methods section. Note the different localization of chitin in the mutant in both yeast and hyphal cells. (B) Intracellular localization of CaChs3-GFP on yeast and hyphal cultures. (C) Panel represents numerical analysis of CaChs3-GFP localization in yeast (n = 3, >100 cells counted in each experiment). Note the apparent loss of signal for CaChs3-GFP in the buds of the exomer mutant. (D) CW vital staining of the indicated mutants of S. cerevisiae. Note the partial delocalization of chitin to the bud in the aps1∆ mutants, a result similar to that observed in the Cachs5∆ mutant (A). See Figure S5 in File S1 for additional data on chitin and CaChs3-GFP localization. CW, calcofluor white; WT, wild-type.

Figure 6

Functional characterization of chimeric Chs3 proteins. (A) Calcofluor (CW) resistance promoted by the chimeric proteins Chs3^CaCT-GFP and ^CaNTChs3-GFP in the indicated S. cerevisiae strains compared to the wild-type ScChs3-GFP used as the control. (B) CW vital staining of the same strains as in (A). (C) Intracellular localization of ScChs3-GFP, Chs3^CaCT-GFP, and ^CaNTChs3-GFP in the indicated strains. (D) Quantitative analysis of the images in (C) (n = 4, >100 cells/experiment). Note the reduced arrival of Chs3^CaCT-GFP at the PM, which is restored in in the aps1∆ mutant. In contrast, ^CaNTChs3-GFP arrives partially at the PM even in the chs5∆ mutant. See Figure S6 in File S1 and Materials and Methods for details on the chimeric constructs.