Control of yeast filamentous-form growth by modules in an integrated molecular network

Abstract

On solid growth media with limiting nitrogen source, diploid budding-yeast cells differentiate from the yeast form to a filamentous, adhesive, and invasive form. Genomic profiles of mRNA levels in Saccharomyces cerevisiae yeast-form and filamentous-form cells were compared. Disparate data types, including genes implicated by expression change, filamentation genes known previously through a phenotype, protein-protein interaction data, and protein-metabolite interaction data were integrated as the nodes and edges of a filamentation-network graph. Application of a network-clustering method revealed 47 clusters in the data. The correspondence of the clusters to modules is supported by significant coordinated expression change among cluster co-member genes, and the quantitative identification of collective functions controlling cell properties. The modular abstraction of the filamentation network enables the association of filamentous-form cell properties with the activation or repression of specific biological processes, and suggests hypotheses. A module-derived hypothesis was tested. It was found that the 26S proteasome regulates filamentous-form growth.

Figure 1

Filamentous growth response of yeast cells. (A) Wild-type yeast-form cells grown in SHAD liquid medium. (B) Wild-type filamentous-form cells grown for 10 h on SLAD agar medium.

Figure 2

Integrated filamentation network. The filamentation network includes proteins (rectangular nodes) implicated in filamentous growth by expression profiling or known phenotypes, and metabolites (triangular nodes) that are either substrates or products of filamentation-protein enzymes. Not shown are filamentation proteins with neither a protein–metabolite interaction nor a protein–protein interaction with another filamentation protein. Blue edges indicate protein–protein interactions. Green edges indicate protein–metabolite interactions. Each gene node is colored based on its expression log-ratio. Shades of red indicate higher expression in the filamentous form relative to the yeast form; shades of blue indicate the opposite response; white indicates no difference.

Figure 3

Modular abstraction of the filamentation network. Network clusters are abstracted as circular “module nodes.” Otherwise, colors and shapes represent the same as in Figure 2. The area of each module node is proportional to the number of member molecules. The color of each module node reflects the average expression log-ratio of member genes. Each module node is automatically assigned the name of the member node of highest intracluster degree (the highest number of interactions with cluster co-members); most are proteins, some are metabolites. See Supplemental Table 3 for a list of cluster members.

Figure 4

RPN12, GRR1, and CDC28 modules and their components. Modules (A), and their respective components (B) with collective functions in cell-cycle control and ubiquitin-dependent proteolysis are shown. Graphic representations are as in Figures 2 and 3.

Figure 5

rpn4Δ mutants show Cln1-dependent hyperelongation, and cell type-independent agar adhesion. (A) Diploid wild-type, rpn4Δ, cln1Δ, and rpn4Δ cln1Δ strains were grown on SLAD agar plates and photographed after 9 h. (B) Patches of strains of the indicated cell types and genotypes were subjected to a wash-off assay of adhesion. The plate was imaged before and after washing with water.

Figure 6

Stabilization of Cln1 protein in rpn4Δ mutants. (A) Northern blot analysis of total RNA from wild-type and rpn4Δ strains grown on SLAD plates, and a cln1Δ strain. The blot was probed consecutively with probes for CLN1 and RPN12. The asterisk in the CLN1 blot indicates a cross-hybridizing band that also serves as a loading control. (B) Western blot analysis of Cln1 protein in diploid wild-type and rpn4Δ strains carrying HA-tagged CLN1, and a no-tag wild-type control strain. Protein extracts were prepared from cells grown for 10 h on SLAD agar plates. Pgk1 protein levels served as a loading control. (C) Cln1-HA protein was immunoprecipitated from an rpn4Δ strain. Aliquots of the immunoprecipitate were incubated with calf-intestine phosphatase (CIP), or without CIP, and analyzed by Western blotting. (D) myc-tagged Cln1 protein was immunoprecipitated in diploid wild-type and rpn4Δ strains, and a no-tag control strain. All strains had a multicopy plasmid expressing HA-tagged ubiquitin. Immunoprecipitates were analyzed by gel electrophoresis and immunoblotting with anti-HA antibody to detect ubiquitin conjugates. The blot membrane was stripped and reprobed with anti-myc antibodies to detect the immunoprecipitated Cln1.