Arabidopsis eIF3e (INT-6) Associates with Both eIF3c and the COP9 Signalosome Subunit CSN7

Avital Yahalom,Tae-Houn Kim,Eitan Winter,Baruch Karniol,Albrecht G Von Arnim,Daniel A Chamovitz

doi:10.1074/jbc.m006721200

Avital Yahalom, Tae-Houn Kim + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m006721200

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Abstract

The Arabidopsis COP9 signalosome is a multisubunit repressor of photomorphogenesis that is conserved among eukaryotes. This complex may have a general role in development. As a step in dissecting the biochemical mode of action of the COP9 signalosome, we determined the sequence of proteins that copurify with this complex. Here we describe the association between components of the COP9 signalosome (CSN1, CSN7, and CSN8) and two subunits of eukaryotic translation initiation factor 3 (eIF3), eIF3e (p48, known also as INT-6) and eIF3c (p105). To obtain a biochemical marker for Arabidopsis eIF3, we cloned the Arabidopsis ortholog of the eIF3 subunit eIF3b (PRT1). eIF3e coimmunoprecipitated with CSN7, and eIF3c coimmunoprecipitated with eIF3e, eIF3b, CSN8, and CSN1. eIF3e directly interacted with CSN7 and eIF3c. However, eIF3e and eIF3b cofractionated by gel filtration chromatography in a complex that was larger than the COP9 signalosome. Whereas eIF3, as detected through eIF3b, localized solely to the cytoplasm, eIF3e, like CSN7, was also found in the nucleus. This suggests that eIF3e and eIF3c are probably components of multiple complexes and that eIF3e and eIF3c associate with subunits of the COP9 signalosome, even though they are not components of the COP9 signalosome core complex. This interaction may allow for translational control by the COP9 signalosome.

Highlights

Light is the major environmental signal regulating plant development
To obtain a biochemical marker for Arabidopsis eukaryotic translation initiation factor 3 (eIF3), we cloned the Arabidopsis ortholog of the eIF3 subunit eIF3b (PRT1). eIF3e coimmunoprecipitated with CSN7, and eIF3c coimmunoprecipitated with eIF3e, eIF3b, CSN8, and CSN1. eIF3e directly interacted with CSN7 and eIF3c
We have previously shown that Arabidopsis eIF3c is highly conserved with the human eIF3c subunit and that eIF3c associates in yeast with the COP9 signalosome components CSN1 and CSN8, probably not as a core component of the COP9 signalosome [29]

Summary

EXPERIMENTAL PROCEDURES

Plant Materials and Growth Conditions—Wild type plants are in the Arabidopsis thaliana Columbia background. Isolation and Cloning of Arabidopsis eIF3e and eIF3b—Internal peptide sequences of the cauliflower COP9 signalosome-associated protein eIF3e were described previously [29]. Following incubation on an Orbiton rotator (Boekel Industries Inc.), the samples were centrifuged for 15 min at 22,000 ϫ g, and the supernatant was transferred to new tubes containing 20 ␮l of protein A-agarose beads (Sigma). For the pull-down assay with CSN7, constant amounts (140 ␮g) of total soluble proteins from E. coli expressing GST-eIF3e were incubated for 1 h at room temperature in PBS buffer containing 1% Triton X-100, with increasing amounts of total soluble proteins from E. coli expressing either His6-CSN7 or control empty pET28a vector (4 ␮g/␮l each). GenBankTM accession numbers for eIF3– eIF3b are from human, T09582; Drosophila, AAF57842.1; S. pombe, Q10425; S. cerevisiae, P06103

Amino acid similarity identity

RESULTS

DISCUSSION