Functional and Structural Studies on Artemia Polypeptide Chain Initiation Factor 2. Cloning and Sequencing of eIF-2α cDNA

Dholakia, Jaydev N.; Reddy, N S; Wahba, Amy

doi:10.1007/978-1-4757-0004-6_34

Cited by 2 publications

(3 citation statements)

References 28 publications

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“…2). These compar- ative data appear to match well with the in vitro casein kinase labeling data for the yeast, Artemia, and human proteins (22,37) and could suggest that casein kinase II phosphorylation of yeast eIF-2a is related to the GCN2-independent phosphorylation of eIF-2o that we see in our in vivo labeling experiment (Fig. 1).…”

Section: Methodssupporting

confidence: 76%

“…2) and is not phosphorylated by the mammalian casein kinase II enzyme (37). Thus, casein kinase II phosphorylation of eIF-2a appears to occur only in S. cerevisiae, and probably Artemia sp., which also contains consensus casein kinase II sequences in the carboxyl end of the protein and is also phosphorylated by this enzyme in vitro (22,37). In contrast, the mechanism of Ser-51 phosphorylation by eIF-2a kinases is conserved in all eukaryotic organisms (15,20 Although casein kinase II would appear not to modify human eIF-2ax, it has been implicated to modify other mammalian translation initiation factors, namely, the ,B subunit of eIF-2 and the 83-kDa subunit of eIF-2B, £ (18,23,24).…”

Section: Discussionmentioning

confidence: 99%

“…Biochemical studies have shown that the a subunit of yeast and Artemia eIF-2 can be phosphorylated by mammalian casein kinase II, whereas the human homolog is not phosphorylated by this kinase in vitro (37). In light of these in vitro data, we compared the amino acid sequences of the yeast (17), Artemia (22), and human (26) (Fig. 2).…”

Section: Methodsmentioning

confidence: 99%

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Casein kinase II mediates multiple phosphorylation of Saccharomyces cerevisiae eIF-2 alpha (encoded by SUI2), which is required for optimal eIF-2 function in S. cerevisiae.

Feng¹,

Yoon²,

Donahue³

1994

Mol. Cell. Biol.

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Previous studies have demonstrated that the a subunit of eukaryotic initiation factor 2 (eIF-2a), encoded by the SUI2 gene in the yeast Saccharomyces cerevisiae, is phosphorylated at Ser-51 by the GCN2 kinase in response to general amino acid control. Here we describe that yeast eIF-2a is a constitutively phosphorylated protein species that is multiply phosphorylated by a GCN2-independent mechanism. 32p; labeling and isoelectric focusing analysis of a SUI2+ Agcn2 strain identifies eIF-2ot as radiolabeled and a single isoelectric protein species. Treatment of SUI2+ Agcn2 strain extracts with phosphatase results in the identification of three additional isoelectric forms of eIF-2a that correspond to the stepwise removal of three phosphates from the protein. These data strongly support the conclusion that casein kinase II directly phosphorylates eIF-2oa at one or all of these Ser amino acids in vivo. Although substitution of SUI2 genes mutated at these sites for the wild-type gene have no obvious effect on cell growth, one test that we have used appears to demonstrate that the inability to phosphorylate these sites has a physiological consequence on eIF-2 function in S. cerevisiae. Haploid strains constructed to contain Ser-to-Ala mutations at the consensus casein kinase II sequences in SUI2 in combination with a mutated allele of either the GCN2, GCN3, or GCD7 gene have synthetic growth defects. These genetic data appear to indicate that the modifications that we describe at the carboxyl end of the eIF-2a protein are required for optimal eIF-2 function in S. cerevisiae.Eukaryotic translation initiation factor 2 (eIF-2) has been extensively characterized at the biochemical and genetic levels. Biochemical studies have established eIF-2 to be composed of three nonidentical subunits, cx, 1, and y, that function during the early steps of translation initiation by forming a ternary complex with GTP and the initiator tRNA (reviewed in references 29 and 39). This complex then binds the 40S ribosomal subunit, which in turn binds the 5' end of mRNA. According to the scanning model, this preinitiation complex scans the leader region until the first AUG codon is reached, whereupon translation begins (reviewed in references 33 and 34). Genetic studies from our laboratory have implicated eIF-2 to also play a role in ribosomal recognition of an AUG start codon. By reverting his4-initiator codon mutants, three unlinked genes, suil, sui2, and SUI3, were identified that when mutated act in trans to restore his4 expression (12). Characterization of the SUI2 and SUI3 genes showed that they encoded the a and a subunits of eIF-2, which are 42 and 58% identical in amino acid sequence to the human eIF-2oa and -13 proteins, respectively (17,25). Further analysis demonstrated that suppressor mutations in these genes conferred the ability to the ribosome to initiate translation at a UUG codon in the early his4 coding region by allowing a mismatched base pair interaction between the UUG codon and the initiator tRNA (17,25,63). The mutations ...

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Section: Methodssupporting

confidence: 76%

Section: Discussionmentioning

confidence: 99%

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Casein kinase II mediates multiple phosphorylation of Saccharomyces cerevisiae eIF-2 alpha (encoded by SUI2), which is required for optimal eIF-2 function in S. cerevisiae.

Feng¹,

Yoon²,

Donahue³

1994

Mol. Cell. Biol.

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Casein Kinase II Mediates Multiple Phosphorylation of Saccharomyces cerevisiae eIF-2α (Encoded by SUI2), Which Is Required for Optimal eIF-2 Function in S. cerevisiae

Feng

Yoon

Donahue

1994

Molecular and Cellular Biology

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Previous studies have demonstrated that the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha), encoded by the SUI2 gene in the yeast Saccharomyces cerevisiae, is phosphorylated at Ser-51 by the GCN2 kinase in response to general amino acid control. Here we describe that yeast eIF-2 alpha is a constitutively phosphorylated protein species that is multiply phosphorylated by a GCN2-independent mechanism. 32Pi labeling and isoelectric focusing analysis of a SUI2+ delta gcn2 strain identifies eIF-2 alpha as radiolabeled and a single isoelectric protein species. Treatment of SUI2+ delta gcn2 strain extracts with phosphatase results in the identification of three additional isoelectric forms of eIF-2 alpha that correspond to the stepwise removal of three phosphates from the protein. Mutational analysis of SUI2 coupled with biochemical analysis of eIF-2 alpha maps the sites to the carboxyl region of SUI2 that correspond to Ser residues at amino acid positions 292, 294, and 301 that compose consensus casein kinase II sequences. 32Pi labeling or isoelectric focusing analysis of eIF-2 alpha from conditional casein kinase II mutants indicated that phosphorylation of eIF-2 alpha is abolished or dephosphorylated forms of eIF-2 alpha are detected when these strains are grown at the restrictive growth conditions. Furthermore, yeast casein kinase II phosphorylates recombinant wild-type eIF-2 alpha protein in vitro but does not phosphorylate recombinant eIF-2 alpha that contains Ser-to-Ala mutations at all three consensus casein kinase II sequences. These data strongly support the conclusion that casein kinase II directly phosphorylates eIF-2 alpha at one or all of these Ser amino acids in vivo. Although substitution of SUI2 genes mutated at these sites for the wild-type gene have no obvious effect on cell growth, one test that we have used appears to demonstrate that the inability to phosphorylate these sites has a physiological consequence on eIF-2 function in S. cerevisiae. Haploid strains constructed to contain Ser-to-Ala mutations at the consensus casein kinase II sequences in SUI2 in combination with a mutated allele of either the GCN2, GCN3, or GCD7 gene have synthetic growth defects. These genetic data appear to indicate that the modifications that we describe at the carboxyl end of the eIF-2 alpha protein are required for optimal eIF-2 function in S. cerevisiae.

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Functional and Structural Studies on Artemia Polypeptide Chain Initiation Factor 2. Cloning and Sequencing of eIF-2α cDNA

Cited by 2 publications

References 28 publications

Casein kinase II mediates multiple phosphorylation of Saccharomyces cerevisiae eIF-2 alpha (encoded by SUI2), which is required for optimal eIF-2 function in S. cerevisiae.

Casein kinase II mediates multiple phosphorylation of Saccharomyces cerevisiae eIF-2 alpha (encoded by SUI2), which is required for optimal eIF-2 function in S. cerevisiae.

Casein Kinase II Mediates Multiple Phosphorylation of Saccharomyces cerevisiae eIF-2α (Encoded by SUI2), Which Is Required for Optimal eIF-2 Function in S. cerevisiae

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