Disease relevance of CDC33

• We describe a simple and rapid purification scheme that can yield 2-5 micrograms of a homogenous and active preparation of eIF-4E from 1 ml of E. coli culture [1].
• Messenger RNAs encoding chloramphenicol acetyltransferase (CAT) with or without the 5'-leader sequence of tobacco mosaic virus (TMV) RNA were synthesized in vitro and translated in Saccharomyces cerevisiae extracts dependent on eukaryotic initiation factors eIF-4E or eIF-4A [2].
• This failure to bind correlated with resistance or reduced susceptibility, suggesting that interruption of the interaction between VPg and this eIF4E paralog may be necessary, but is not sufficient for potyvirus resistance in vivo [3].
• Effective expression of the active eIF-4E was achieved in the soluble fraction of the insect cell Sf9, which was infected with the recombinant baculovirus [4].

High impact information on CDC33

• It interacts with the small ribosomal subunit interacting protein, eIF3, and the eIF4E/cap-mRNA complex in order to load the ribosome onto mRNA during cap-dependent translation [5].
• Cofolding allosterically enhances association of eIF4E with the cap and is required for maintenance of optimal growth and polysome distributions in vivo [5].
• We find that Pab1p but not the cap binding protein eIF-4E is required for poly(A) tail-dependent translation, and that the Pab1p-poly(A) tail complex functions to recruit the 40S ribosomal subunit to the mRNA [6].
• Using distinct vesicular transport mutants and chlorpromazine, we have identified the eIF2alpha kinase Gcn2p and the eIF4E binding protein Eap1p as major mediators of the translation attenuation response [7].
• Here we report genetic and biochemical evidence that the yeast translation initiation factor eIF4G associates with CBC, and that eIF4E, the eIF4F component that binds both the cap and eIF4G, antagonizes this interaction [8].

Biological context of CDC33

• Another corresponds to CDC33, which codes for the initiation factor 4E or cap binding protein [9].
• Sequence analysis of a 9873 bp fragment of the left arm of yeast chromosome XV that contains the ARG8 and CDC33 genes, a putative riboflavin synthase beta chain gene, and four new open reading frames [9].
• In contrast, uncapped, poly(A)-deficient mRNA is translated poorly in yeast extracts, in part because of the absence of eIF4E and Pab1p binding sites on the mRNA [10].
• mRNA translation in crude extracts from the yeast Saccharomyces cerevisiae is stimulated by the cap structure and the poly(A) tail through the binding of the cap-binding protein eukaryotic translation initiation factor 4E (eIF4E) and the poly(A) tail-binding protein Pab1p [10].
• We propose that regulation of translation initiation by Cdc33 plays a pivotal role in the activation of Start and cell cycle progression in budding yeast [11].

Anatomical context of CDC33

• Furthermore, in cdc33 mutant cells, the size of polysomes containing SSA1-lacZ mRNA was unaffected, while polysomes containing other specific mRNAs were smaller [12].
• A rate-limiting step during translation initiation in eukaryotic cells involves binding of the initiation factor eIF4E to the 7-methylguanosine-containing cap of mRNAs [13].
• Crosses between rnr2-68(ts) and mutant eIF4E (cdc33-1(ts)) engendered conditional synthetic lethality, with extreme sensitivity to hydroxyurea and the microtubule depolymerizing agent, benomyl [14].
• Electroporation of yeast spheroplasts with in vitro synthesized mRNA allowed us to characterize the ability of eIF-4E mutant strains to distinguish between capped and uncapped mRNAs in vivo [15].
• Other phosphorylated proteins include mRNA cap-binding protein from mammalian erythrocytes and yeast, a glucocorticoid receptor protein, and a preparation of the guanine nucleotide-binding proteins Gi and Go from brain [16].

Associations of CDC33 with chemical compounds

• Transcription from the GAL1 promoter (and consequently the production of wild-type eIF-4E) was then shut off by plating these cells on glucose-containing medium [17].
• Comparison of the cdc33 mutation with other mutations affecting eIF-4E function supports the view that tryptophane residues and their flanking regions are involved in cap binding activity of eIF-4E [18].
• Measurements of interaction of 7-methyl-GTP eIF4E from S. cerevisiae were performed by means of two methods: Isothermal Titration Calorimetry (ITC) and fluorescence titration [19].

Physical interactions of CDC33

• Specifically, we find that mutations which weaken the eIF4E binding site on the yeast eIF4G proteins Tif4631p and Tif4632p lead to temperature-sensitive growth in vivo and the stimulation of uncapped-mRNA translation in vitro [10].
• High-copy expression of a mutant eIF4G defective for eIF4E binding resulted in a dominant negative phenotype in an xrn1 mutant, indicating the importance of this interaction in an xrn1 mutant [20].
• NMD can trigger decay during any round of translation and can target Cbc-bound or eIF-4E-bound transcripts [21].
• The cap binding protein eIF-4E copurifies with Ebs1p in the absence of RNA, suggesting that the two proteins interact in vivo [22].
• In yeast, EAP1 encodes a protein that binds eIF4E and inhibits cap-dependent translation in vitro [11] [13].

Regulatory relationships of CDC33

• In addition, we demonstrate that in vivo a temperature-sensitive allele of eIF4E (cdc33-42) suppressed the decapping defect of a partial loss-of-function allele of DCP1 [23].
• The mutagenized gene was reintroduced on a plasmid into S. cerevisiae cells having their only wild-type eIF-4E gene on a plasmid under the control of the regulatable GAL1 promoter [17].
• This suggests that serum stimulates the interaction between eIF4G and PABP by a distinct mechanism that is independent of both the mTOR pathway and the enhanced association of eIF4G with eIF4E [24].

Other interactions of CDC33

• These experiments indicated that eIF4E is an inhibitor of Dcp1p in vitro due to its ability to bind the 5' cap structure [23].
• This lengthened 5'-untranslated region likely reduces translation efficiency and down-regulates CLN3 protein synthesis, thereby correcting for the excess translation promoted by elevated Cdc33 [11].
• Thus, the inhibition of mRNA turnover by blocking Xrn1p function does not suppress the lethality of defects upstream in the turnover pathway but it does enhance the requirement for (7)mG caps and for proper formation of the eIF4E/eIF4G cap recognition complex [20].
• A cdc33-1 strain expressing either stable Cln3p (Cln3-1p) or a hybrid UBI4 5'-CLN3 mRNA, whose translation displays decreased dependence on eIF4E, arrested randomly in the cell cycle [25].
• Another allele of CDC33, cdc33-1, along with mutations in CEG1, encoding the nuclear guanylyltransferase, were also synthetic lethal with xrn1Delta, whereas mutations in PRT1, encoding a subunit of eIF3, were not [20].

Analytical, diagnostic and therapeutic context of CDC33

• We report here the molecular cloning of the CDC33 gene by complementation of the cdc33-1 thermosensitive mutant [26].
• Here, we demonstrate the reconstitution of an eIF4E/eIF4G/Pab1p complex with recombinant proteins, and show by atomic force microscopy that the complex can circularize capped, polyadenylated RNA [27].
• Circular dichroism and fluorescence studies on protein synthesis initiation factor eIF-4E and two mutant forms from the yeast Saccharomyces cerevisiae [28].
• Gel chromatography and immunoadsorption experiments with a monoclonal anti-eIF-4E antibody coupled to protein G-Sepharose show that both p150 and p20 bind independently of each other to eIF-4E [29].
• Supporting this, Western blot analysis shows that far more eIF-4E protein is present in cdc33 yeast cells expressing the RpS15a gene than lacking it [30].

References