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

Abstract

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 ...

Identification of a Translation Initiation Factor 3 (eIF3) Core Complex, Conserved in Yeast and Mammals, That Interacts with eIF5

“…detected in a comparable extract prepared from strain Ad (24). Thus, expression of the Ubi-Nip1p fusion was repressed and the preexisting fusion protein was degraded in cells grown on glucose-containing medium.…”

“…The construction of strain NIP1KR4R1, containing a fusion between ubiquitin and Nip1p expressed under the control of the GAL10 promoter, from strain Ad (MATa NIP1 ade2 his3 leu2 trp1 ura3 can1 rho ϩ L-0 M-0) is described elsewhere (24).…”

Poly(A)-Binding Protein Acts in Translation Termination via Eukaryotic Release Factor 3 Interaction and Does Not Influence [PSI+] Propagation

“…Moreover, a nonsense mutation in yeast can be phenotypically suppressed with paromomycin (74,87), which also increases the efficiency of [PSI ϩ ]-dependent suppression (75). It was found previously that a mutation in a gene encoding a translation initiation factor could also cause paromomycin sensitivity, and it was proposed elsewhere that paromomycin could affect the function of any protein involved in the decoding process (40).…”

A 110-Kilodalton Subunit of Translation Initiation Factor eIF3 and an Associated 135-kilodalton Protein Are Encoded by theSaccharomyces cerevisiae TIF32 and TIF31Genes

“…This may represent a core complex of eIF3, as it possesses activity in vitro. In the IMAC and immunoprecipitation experiments reported here, tagged p33, p39, or p110 lead to copurification of the same 5 subunits, except for p93 which is known to be unstable in strains derived from W303 (17) and presumably is degraded in the lysates examined. It is noteworthy that only the 5 core subunits have homologs in mammalian eIF3 (see below).…”

“…The genes encoding the p39 (TIF34) and p33 (TIF35) subunits have been cloned and characterized more recently (14,15). The p29 subunit was shown to be a degradation product of p39 (14), whereas a protein of 93 kDa has been identified as an eIF3 subunit (p93) encoded by NIP1 (16,17). Nip1p (p93) is not present in our preparation described above, apparently due to proteolysis during purification.…”