Nucleic Acids Research, 2010, Vol. 38, No. 22 8045 Figure 3. Functional classiﬁcation of regulated mRNAs. Yeast ‘GO Slim’ summary of signiﬁcantly enriched and under-represented gene Ontology classes calculated using the hypergeometric distribution (performed at www.yeastgenome.org), see Supplementary Table S10 for more details including all calculated P-values. Bold text indicates most over-represented classes (P < 0.00001) in one or more experiment. play a role repressing translation of these mRNAs in the mother cell, but that localized transcripts could become translatable. Bud-localized activation of ASH1 mRNA translation has been studied directly and two alternative hypotheses have been proposed to account for its transla- tional repression involving Khd1p, eIF4G and the kinase Yck1p (32) or involving eIF5B and Puf6p (33). Our data is consistent with and suggestive that the yeast 4E-BPs could play a role in spatial regulation of translation for speciﬁc transcripts in yeast but elucidation of molecular mechan- isms is beyond the scope of this article. Nitrogen and amino acid metabolism defects Filamentous or pseudohyphal growth on poor nitrogen sources in 1278b yeast strains requires reprogramming of cellular growth. We recently showed that each 4E-BP was required for this response (10) highlighting a role for translational control in the response to nitrogen limita- tion. Others found that both CLN1 and STE12 translation is induced following induction of ﬁlamentous growth on poor nitrogen sources in 1278b yeast (34). Our arrays were performed on S288c derived cells that do not undergo this developmental switch because they lack the FLO8 transcription factor required for induction of some developmentally regulated genes (35), and on cells grown in a nitrogen sufﬁcient medium. Nevertheless, both CLN1 and STE12 are translationally up-regulated in our arrays, (CLN1 by both factor deletions and STE12 by eap1D) suggesting that 4E-BP mediated repression of certain ﬁla- mentous growth genes may occur during growth on rich media. Our array experiments also predicted that both caf20D and eap1D cells would have unbalanced nitrogen and amino acid metabolism gene expression due to altered translation of ammonia and amino acid transporters and metabolic enzymes. A large number of genes in this group are down-regulated in eap1D cells (Supplementary Table S8). To assess if these observations had phenotypic conse- quences, auxotrophic markers in each strain were comple- mented and cells were plated on a variety of media and growth scored. A summary of the results is shown in Figure 4. When grown on standard complete media con- taining ammonium sulphate and a mixture of all amino acids all cells grew well. The eap1D cells in particular grew very poorly on less complex media containing sole nitrogen sources. Measurements of maximum doubling times in liquid cultures conﬁrmed that caf20D also showed defects with some nitrogen sources albeit to a lesser extent than seen with eap1D (Table 1). As the glu- tamate/aspartate transporter DIP5 (36) transcript levels were 2-fold reduced in eap1D cells (Supplementary Table S4) it was important to ascertain whether these phenotypes were dependent upon translational controls. We found that the wild-type EAP1 plasmid rescued