The 39-Kilodalton Subunit of Eukaryotic Translation Initiation Factor 3 Is Essential for the Complex’s Integrity and for Cell Viability in <i>Saccharomyces cerevisiae</i>

Nip1p is an essential Saccharomyces cerevisiae protein that was identified in a screen for temperature conditional (ts) mutants exhibiting defects in nuclear transport. New results indicate that Nip1p has a primary role in translation initiation. Polysome profiles indicate that cells depleted of Nip1p and nip1-1 cells are defective in translation initiation, a conclusion that is supported by a reduced rate of protein synthesis in Nip1p-depleted cells. Nip1p cosediments with free 40 S ribosomal subunits and polysomal preinitiation complexes, but not with free or elongating 80 S ribosomes or 60 S subunits. Nip1p can be isolated in an about 670-kDa complex containing polyhistidine-tagged Prt1p, a subunit of translation initiation factor 3, by binding to Ni 2؉ -NTA-agarose beads in a manner completely dependent on the tagged form of Prt1p. The nip1-1 ts growth defect was suppressed by the deletion of the ribosomal protein, RPL46. Also, nip1-1 mutant cells are hypersensitive to paromomycin. These results suggest that Nip1p is a subunit of eukaryotic initiation factor 3 required for efficient translation initiation.Translation initiation can be considered to begin with the dissociation of 80 S ribosomes into free 40 S and 60 S subunits which then reassociate with mRNAs in a highly regulated and complex process. Initiation of cap-dependent translation in eukaryotes begins with the binding of a 43 S preinitiation complex to the 5Ј cap of the mRNA. The 43 S ribosome migrates down the mRNA to the translation start site where it is joined by a 60 S ribosomal subunit to complete the assembly of an apparatus capable of accurately forming the first peptide bond. Translation initiation is mediated by at least 10 translation initiation factors, many of which contain multiple subunits that are conserved between yeast and mammals (reviewed in Refs. 1). eIF3 1 is the most complex initiation factor and the one which is least understood with regard to both composition and function. Human eIF3, which is composed of at least nine distinct subunits (2-5), stimulates multiple steps of translation initiation, including the dissociation of 80 S ribosomes, stabilizing tRNA i Met binding to 40 S ribosomal subunits, and binding of mRNA to 40 S subunits (1). A yeast eIF3 complex was purified on the basis of replacing mammalian eIF3 in an in vitro translation initiation assay (6). This complex contains eight subunits of masses 16,21,29,33,39,62, 90, and 135 kDa. The 16-, 39-, 62-, and 90-kDa subunits were identified as Sui1p (7, 8), Tif34p (9) Gcd10p (10), and Prt1p (11), respectively. Yeast Prt1p is 36% identical to the 116-kDa subunit of human eIF3, and Tif34p, a WD repeat protein, is 46% identical to the p36 subunit of human eIF3. Tif34p is required for cell cycle progression and mating as well as translation initiation (12). However, Gcd10p and Sui1p do not show strong similarities to any human eIF3 subunits (13). p33 was shown to interact with Tif34p and Prt1p by two-hybrid analysis and co-immunoprecipitation. It has a RNA-binding domain ...

Section: Cells Depleted Of Nip1p Have a Reduced Rate Of Protein Synthmentioning

confidence: 54%

Nip1p Associates with 40 S Ribosomes and the Prt1p Subunit of Eukaryotic Initiation Factor 3 and Is Required for Efficient Translation Initiation

Greenberg¹,

Phan²,

Gu³

et al. 1998

“…3B, lanes 4 and 5) sedimented in fractions just preceding the 40 S ribosome, suggesting that these proteins are present in high molecular mass complexes. Immunoblotting of the S. cerevisiae fractions with antibodies against the budding yeast eIF3b (PRT1) supports the notion that the high molecular mass complex containing HA-TIF34 at fractions 4 -5 is the eIF3 complex of ϳ600 kDa (17,18,28). As expected, the S. cerevisiae eIF2 complex of 124 kDa sedimented in fractions closer to the top of the gradient, with a peak in fraction 3, whereas the bulk of eIF1 (13 kDa) peaked in fraction 2 (Fig.…”

Section: Int6 Is Part Of a High Molecular Mass Complexmentioning

confidence: 79%

Fission Yeast Homolog of Murine Int-6 Protein, Encoded by Mouse Mammary Tumor Virus Integration Site, Is Associated with the Conserved Core Subunits of Eukaryotic Translation Initiation Factor 3

Akiyoshi

Clayton

Phan

et al. 2001

The murine int-6 locus, identified as a frequent integration site of mouse mammary tumor viruses, encodes the 48-kDa eIF3e subunit of translation initiation factor eIF3. Previous studies indicated that the catalytically active core of budding yeast eIF3 consists of five subunits, all conserved in eukaryotes, but does not contain a protein closely related to eIF3e/Int-6. Whereas the budding yeast genome does not encode a protein closely related to murine Int-6, fission yeast does encode an Int-6 ortholog, designated here Int6. We found that fission yeast Int6/eIF3e is a cytoplasmic protein associated with 40 S ribosomes. FLAG epitope-tagged Tif35, a putative core eIF3g subunit, copurified with Int6 and all five orthologs of core eIF3 subunits. An int6 deletion (int6⌬) mutant was viable but grew slowly in minimal medium. This slow growth phenotype was accompanied by a reduction in the amount of polyribosomes engaged in translation and was complemented by expression of human Int-6 protein. These findings support the idea that human and Schizosaccharomyces pombe Int-6 homologs are involved in translation. Interestingly, haploid int6⌬ cells showed unequal nuclear partitioning, possibly because of a defect in tubulin function, and diploid int6⌬ cells formed abnormal spores. We propose that Int6 is not an essential subunit of eIF3 but might be involved in regulating the activity of eIF3 for translation of specific mRNAs in S. pombe.

“…It will therefore be important to identify with which other protein(s) TRIP-1 interacts when associated with the type II receptor. TRIP-1 has recently been identified as a component of the human translation initiation factor complex eIF3 (33), and the yeast TRIP-1 homologue TIF34 has also been found as a subunit of yeast eIF3 (34,35). Accordingly, sequential immunoprecipitation analyses using TRIP-1 antibodies and an antiserum against the eIF3 complex revealed an interaction of TRIP-1 with components of the complex.…”

Section: Discussionmentioning

confidence: 99%

The Type II Transforming Growth Factor (TGF)-β Receptor-interacting Protein TRIP-1 Acts as a Modulator of the TGF-β Response

Choy

Derynck

1998

116

The transforming growth factor-␤ (TGF-␤) receptor interacting protein TRIP-1 was originally identified as a WD40 repeat-containing protein that has the ability to associate with the TGF-␤ type II receptor and is phosphorylated by it (1). However, its function was not known. We now show that TRIP-1 expression represses the ability of TGF-␤ to induce transcription from the plasminogen activator inhibitor-1 promoter, a common reporter of the TGF-␤-induced gene expression response, but does not affect the ability of TGF-␤ to inhibit cyclin A transcription. TRIP-1 can also inhibit the plasminogen activator inhibitor-1 expression induced by Smads as well as activated TGF-␤ type I receptors. Its inhibitory effect is exerted by a combination of receptordependent and receptor-independent mechanisms. Deletion mutational analysis revealed that two distinct regions, which do not contain recognizable WD40 repeats, are required for the ability of TRIP-1 to inhibit the gene expression response. Expression of other segments of TRIP-1 increased the TGF-␤-induced gene expression response and therefore may exert a dominant negative phenotype. We conclude that TRIP-1 acts as a modulator of the TGF-␤ response. Transforming growth factor-␤ (TGF-␤)1 is the prototype member of a superfamily of growth factors, which have many roles in growth regulation, wound healing, immunity, and development. Most notably, TGF-␤ is a potent growth inhibitor for many cell types and induces a variety of extracellular matrix proteins and adhesion receptors (2). The current model for TGF-␤ signaling invokes the binding of ligand to the type II TGF-␤ receptor, which results in the recruitment, phosphorylation, and concomitant activation of type I TGF-␤ receptors. The activated type I receptor then phosphorylates Smad2 or Smad3, which is then released from the receptor and forms a complex with Smad4. This complex then translocates to the nucleus where it interacts with transcription factors and regulates gene expression (3).Although Smads clearly act as effectors of TGF-␤ receptor signaling, several lines of evidence indicate that other factors are probably involved in TGF-␤ signal transduction. The ability of a dominant negative type II receptor to inhibit TGF-␤-mediated growth inhibition but not extracellular matrix production suggests that the pathways leading to these effects of TGF-␤ are distinct and separable (4) and may be differentially regulated via quantitatively different thresholds of signaling (5). Consistent with this notion, specific residues on the type I (6) or type II (7) receptor have been identified that are dispensable for TGF-␤ stimulation of extracellular matrix production but required for the antimitogenic effect. The Smads have been found to mediate both the extracellular matrix and the antimitogenic signaling pathways (3,8,9), and no evidence yet presented has indicated a role for Smads in differentially modulating distinct TGF-␤ signaling pathways. Thus, although the basis for distinction between these pathways is not well underst...