An interactional network of genes involved in chitin synthesis in Saccharomyces cerevisiae

Abstract

Background: In S. cerevisiae the beta-1,4-linked N-acetylglucosamine polymer, chitin, is synthesized by a family of 3 specialized but interacting chitin synthases encoded by CHS1, CHS2 and CHS3. Chs2p makes chitin in the primary septum, while Chs3p makes chitin in the lateral cell wall and in the bud neck, and can partially compensate for the lack of Chs2p. Chs3p requires a pathway of Bni4p, Chs4p, Chs5p, Chs6p and Chs7p for its localization and activity. Chs1p is thought to have a septum repair function after cell separation. To further explore interactions in the chitin synthase family and to find processes buffering chitin synthesis, we compiled a genetic interaction network of genes showing synthetic interactions with CHS1, CHS3 and genes involved in Chs3p localization and function and made a phenotypic analysis of their mutants.

Results: Using deletion mutants in CHS1, CHS3, CHS4, CHS5, CHS6, CHS7 and BNI4 in a synthetic genetic array analysis we assembled a network of 316 interactions among 163 genes. The interaction network with CHS3, CHS4, CHS5, CHS6, CHS7 or BNI4 forms a dense neighborhood, with many genes functioning in cell wall assembly or polarized secretion. Chitin levels were altered in 54 of the mutants in individually deleted genes, indicating a functional relationship between them and chitin synthesis. 32 of these mutants triggered the chitin stress response, with elevated chitin levels and a dependence on CHS3. A large fraction of the CHS1-interaction set was distinct from that of the CHS3 network, indicating broad roles for Chs1p in buffering both Chs2p function and more global cell wall robustness.

Conclusion: Based on their interaction patterns and chitin levels we group interacting mutants into functional categories. Genes interacting with CHS3 are involved in the amelioration of cell wall defects and in septum or bud neck chitin synthesis, and we newly assign a number of genes to these functions. Our genetic analysis of genes not interacting with CHS3 indicate expanded roles for Chs4p, Chs5p and Chs6p in secretory protein trafficking and of Bni4p in bud neck organization.

Figure 1

A network of genetic interactions with BNI4, CHS1, CHS3, CHS4, CHS5, CHS6 and CHS7. (A) Global view of the network. Synthetic interactions with any query gene (diamonds) are depicted as edges joining these to nodes (circles). Nodes whose deletion mutant have a decreased, wild type and increased chitin content are colored in green, gray and red, respectively. For the decreased (green) and increased (red) chitin contents, color intensity is proportional to the magnitude of the change. (B) Venn diagram of the CHS1 interaction set with the CSIII network. The number of genes interacting with CHS1 or with any of the CSIII query genes is indicated. The numbers in parentheses indicate the number of interactions for multiply connected genes. Genes showing 2 or more interactions are grouped in green or red ovals, respectively.

Figure 2

Analysis of the CSIII network. (A) Grouping genes of the CSIII network in functional categories. Genes belonging to the "core" group (CHS3 plus at least one other) are underlined. The proportions of the functional categories in the CSIII network and in the "core" group are represented in the outer and the inner pies, respectively. (B) Interactions among the "core" group. Color coding for nodes is as in (A).

Figure 3

Grouping deletion mutants with an altered chitin content according to their interaction pattern. (A) Chitin levels, expressed in nmole GlcNAc/mg dry weight, in wild type, group 1 and query mutants. (B) Chitin levels in wild type and group 2 mutants. Note the different scales in (A) and (B). Hypersensitivity, resistance, wild type and not determined sensitivity to Calcofluor are indicated by black, open, gray and hatched bars, respectively.

Figure 4

Grouping deletion mutants with wild type chitin content according to their interaction pattern. (A) and (B) group 3 and 4 mutants, respectively. Hypersensitivity, resistance, wild type and not determined sensitivity to Calcofluor are indicated by bold, open, gray and underlined characters, respectively.

Figure 5

Comparative synthetic interaction patterns of CHS4 and SHC1. Synthetic interactions with CHS4 or SHC1 are depicted as connections between these nodes and their respective partners (black and gray nodes, respectively).

Figure 6

Functional integration of CHS1- and CHS3-interaction sets. CHS1- and CHS3-interacting genes were grouped according to their effects on chitin synthesis. The Venn diagram shows the distinct and overlapping sets for each functional category.