Pull-down distributions. Distributions of log2 ratios obtained by averaging all independent microarray hybridization experiments for each strain, wild type (SC0000), and the TAP-tagged versions of Whi3 (SC3304), Whi4 (SC3411), Dpm1 (SC1622), and Sfb3 (SC2482) are shown. Values are plotted scaled to the population standard deviations. The distribution of selected Whi3 targets is shown in red, and the position of CLN3 is marked by an arrow.

Whi3 as a determinant of protein fate. The diagram shows a speculative model summarizing data obtained. Whi3 would bind ER-associated ribonucleoparticles containing specific mRNA targets enriched in GCAU clusters. For some of them, including CLN3, Whi3 would be necessary for an efficient retention at the ER of the encoded products. Alternatively, Whi3-mediated recruitment of many other mRNAs would localize their translation at the cytosolic face of the ER and/or contribute to an efficient translocation across the ER of those encoded proteins destined to the exocytic pathway.

Whi3-associated mRNAs are enriched in clusters of the GCAU tetranucleotide. A, to quantify the enrichment in GCAU clusters, for each GCAU sequence in the mRNA, the distance to a third consecutive hit was measured. Graphs show the frequency of these distances in base pairs (which contain four consecutive GCAU hits) among the Whi3 transcripts when compared with the same number of transcripts from mock pull down (Ctr IP) and the total genome. B, same as in A for the CGUA tetranucleotide sequence. C, the graph shows median and confidence limits for the median for each distribution plotted in A.

Identification of Whi3-associated mRNAs. A, TreeView generated heat map showing the top 50 candidate targets for Whi3. Genes are in rows; independent replicate IPs are in columns. Yellow denotes enrichment in the IP relative to the input RNA. Log2 ratio values are plotted. B, correlation analysis of all the arrays performed in this study. The software Cluster 3.0 (51) was used to cluster arrays using an uncentered Pearson correlation similarity metric and average linkage. Other metrics gave similar results. Java TreeView was used to generate the tree.

Whi3-deficient cells show cell wall perturbations. A, evaluation of growth sensitivity to Congo Red (CR) and Calcofluor White (CFW) drugs of wild-type (CML128), whi3Δ (CML217), whi4Δ (CML219), whi3Δwhi4Δ (CYC285), and whi3ΔRRM (CYC159) cells 3 days after plating a series of 5-fold diluted spots in rich medium. B, analysis of growth capacities at different temperatures and effects of 0.8 m sorbitol (Sorb) in wild-type (CML128), whi3Δ (CML217), whi4Δ (CML219), whi3Δwhi4Δ (CYC285), slt2Δ (CML399), and whi3Δslt2Δ (CYC287) cells streaked in rich medium. C, Western blot analysis of Slt2 phosphorylation in wild-type (CML128) and whi3Δ (CML217) cells harboring the Yep352-Slt2-3HA plasmid. Cell cultures were exponentially grown in minimal medium at 25 °C. Western blot analysis was performed to detect activated (P-Slt2) and total (Slt2-3HA) amounts of Slt2. P-Kss1, activated Kss1; P-Fus3; activated Fus3.

Characterization of Whi3 targets. A, classification of proteins encoded for by the Whi3-associated mRNAs based on subcellular localization information gathered from MIPS (34) and SGD (35) data bases. The percentage of each compartment is indicated inside the chart. The percentages of the yeast proteome were obtained from Kumar et al. (36). The significance in the enrichment (p value) of the exocytosis network was obtained from the Funspec data base (52) B, genes in the Whi3 data set were classified by cellular component terms (GO component) gathered from SGD data base (see supplemental Table S1). C, analysis of the predicted transmembrane domains in the Whi3 data set by the Simple Modular Architecture Research Tool (SMART) domain data base (52, 53) (see supplemental Table S1, funspec data). D, Whi3 targets encode for proteins involved in transport. Genes in the Whi3 data set were classified by biological function terms (GO function) gathered from the SGD data base (see supplemental Table S1). B, C, and D, the comparative enrichment in the various categories between the Whi3 data set (gray columns) and the entire yeast genome (black columns) is depicted together with the significance values (p values).

Mutation of the GCAU clusters in the CLN3 mRNA reduces the interaction with Whi3. A, diagram representing the distribution of the 14 GCAU sequences (dashes) in the CLN3 mRNA that were mutated to obtain the CLN3^mGCAU allele. UTR, untranslated region; ORF, open reading frame. B, a cln3Δ Whi3-TAP (CYC124) strain transformed with plasmids expressing wild-type (pCLN3) and mutant (pCLN3^mGCAU) mRNAs was used for microarray analysis of Whi3-TAP pull downs. CLN3 ratios were made relative to the top hundred targets of Whi3 obtained from experiments shown in Fig. 1. Two independent experiments for the wild-type chromosomal CLN3 (Chr CLN3) are also shown. The values are obtained by averaging the four CLN3 spots on each array. Error bars represent confidence limits of the mean. C, cln3Δ (CML211) cells expressing 3HA-tagged wild-type (pCLN3) and mutant (pCLN3^mGCAU) constructs on centromeric vectors were grown exponentially and analyzed by immunofluorescence with an anti-HA antibody. Nuclei were detected by 4′,6-diamidino-2-phenylindole staining. Bar, 5 μm. D, the percentage of cells (n = 300) with Cln3-3HA nuclear/cytoplasmic ratios higher than 2; confidence limits (α = 0.01) are plotted. E, protein extracts of cells corresponding to C were analyzed by Western blot with an anti-HA antibody. The asterisk indicates an unspecific band. F, cell volume distributions of cells corresponding to C. Wild-type (CML128) and whi3Δ mutant (CML217) cells growing under the same conditions were used as internal controls.