Evidence that GCN1 and GCN20, Translational Regulators of GCN4 , Function on Elongating Ribosomes in Activation of eIF2α Kinase GCN2

Aug 1, 1997Molecular and cellular biology

Evidence that two protein regulators work with active ribosomes to activate a stress-response enzyme

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Abstract

The C-terminal 84% of GCN20 is dispensable for complex formation with GCN1 and for stimulating GCN2 kinase function.

Phosphorylation of translation initiation factor eIF2 by GCN2 increases translation of GCN4 in amino acid-starved yeast cells.
GCN1 and GCN20 form a protein complex that is required for stimulating GCN2 kinase activity during starvation.
Indirect immunofluorescence shows GCN1 is localized in the cytoplasm, with no clear association with membranes.
A fraction of GCN1 and GCN20 associates with polysomes and 80S ribosomes, indicating a potential role in translation.
The N-terminal 15 to 25% of GCN20, essential for its regulatory function, interacts with a segment of GCN1 similar to translation elongation factor 3.
Similar mechanisms for detecting uncharged tRNA on translating ribosomes may exist in both yeast and human cells.

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In the yeast Saccharomyces cerevisiae, phosphorylation of translation initiation factor eIF2 by protein kinase GCN2 leads to increased translation of the transcriptional activator GCN4 in amino acid-starved cells. The GCN1 and GCN20 proteins are components of a protein complex required for the stimulation of GCN2 kinase activity under starvation conditions. GCN20 is a member of the ATP-binding cassette (ABC) family, most of the members of which function as membrane-bound transporters, raising the possibility that the GCN1/GCN20 complex regulates GCN2 indirectly as an amino acid transporter. At odds with this idea, indirect immunofluorescence revealed cytoplasmic localization of GCN1 and no obvious association with plasma or vacuolar membranes. In addition, a fraction of GCN1 and GCN20 cosedimented with polysomes and 80S ribosomes, and the ribosome association of GCN20 was largely dependent on GCN1. The C-terminal 84% of GCN20 containing the ABCs was found to be dispensable for complex formation with GCN1 and for the stimulation of GCN2 kinase function. Because ABCs provide the energy-coupling mechanism for ABC transporters, these results also contradict the idea that GCN20 regulates GCN2 as an amino acid transporter. The N-terminal 15 to 25% of GCN20, which is critically required for its regulatory function, was found to interact with an internal segment of GCN1 similar in sequence to translation elongation factor 3 (EF3). Based on these findings, we propose that GCN1 performs an EF3-related function in facilitating the activation of GCN2 by uncharged tRNA on translating ribosomes. The physical interaction between GCN20 and the EF3-like domain in GCN1 could allow for modulation of GCN1 activity, and the ABC domains in GCN20 may be involved in this regulatory function. A human homolog of GCN1 has been identified, and the portion of this protein most highly conserved with yeast GCN1 has sequence similarity to EF3. Thus, similar mechanisms for the detection of uncharged tRNA on translating ribosomes may operate in yeast and human cells.

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Evidence that GCN1 and GCN20, Translational Regulators of GCN4 , Function on Elongating Ribosomes in Activation of eIF2α Kinase GCN2

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