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The identification of the activator of heat shock protein 90 (Hsp90) ATPase's (Aha1) protein-protein interaction (PPI) network will provide critical insights into the relationship of Aha1 with multi-molecular complexes and shed light onto Aha1's interconnections with Hsp90-regulated biological functions. Flag-tagged Aha1 was over-expressed in HeLa cells and isolated by anti-Flag affinity pull downs, followed by trypsin digestion and identification co-adsorbing proteins by liquid chromatography-tandem mass spectroscopy (LC-MS/MS). A probability-based identification of Aha1 PPIs was generated from the LC-MS/MS analysis by using a relative quantification strategy, spectral counting (SC). By comparing the SC-based protein levels between Aha1 pull-down samples and negative controls, 164 Aha1-interacting proteins were identified that were quantitatively enriched in the pull-down samples over the controls. The identified Aha1-interacting proteins are involved in a wide number of intracellular bioprocesses, including DNA maintenance, chromatin structure, RNA processing, translation, nucleocytoplasmic and vesicle transport, among others. The interactions of 33 of the identified proteins with Aha1 were further confirmed by Western blotting, demonstrating the reliability of our affinity-purification-coupled quantitative SC-MS strategy. Our proteomic data suggests that Aha1 may participate in diverse biological pathways to facilitate Hsp90 chaperone functions in response to stress.

Eukaryotic initiation factor-2B (eIF-2B) is a guanine nucleotide-exchange protein involved in the recycling of eIF-2 during peptide-chain initiation. Regulation of eIF-2B activity occurs under a wide range of conditions by diverse mechanisms. To better understand the regulation of eIF-2B activity as well as the coordinate expression of its five subunits, we have begun to clone and characterize the cDNAs and genes encoding these proteins. In the present study, complementary and genomic DNAs encoding the epsilon-subunit of rat eIF-2B were cloned and characterized. The cDNA is 2517 bp in length, including a 30 nt poly(A) tail, and recognizes both 2.7 and 3.5 kb mRNA species on Northern blots of rat RNA. The cDNA contains a 2151 bp open reading frame encoding 716 amino acids producing a protein with a predicted molecular mass of 80 kDa. The derived amino acid sequence contains regions identical to three peptides obtained from bovine liver eIF-2B epsilon and is 31% identical to Gcd6, the putative yeast eIF-2B epsilon. Examination of the derived amino acid sequence of rat eIF-2B epsilon reveals phosphorylation site motifs for several protein kinases which have been implicated in regulation of guanine nucleotide exchange activity. The mRNA for eIF-2B epsilon is expressed to a similar extent in most rat tissues examined with the exception of testis, where its expression is approx, three-fold greater. We have also isolated and sequenced the coding and 5'-flanking region of the rat eIF-2B epsilon gene. The 16 exons encoding rat eIF-2B epsilon are contained within 9.5 kb of genomic DNA. Examination of the promoter region of the gene reveals a consensus binding site for the alpha-Pal transcription factor as well as possible cytokine-response elements and binding sites for testis-specific transcription factors.

A rabbit reticulocyte lysate cDNA library was screened with a polyclonal antiserum directed against eukaryotic initiation factor eIF-2B (eIF-2B). A 2508 base pair cDNA (pA1) was isolated and determined to encode the epsilon-subunit of eIF-2B based on the immunoreactivity of the fusion protein expressed from the cDNA in Escherichia coli and the presence of two peptide sequences obtained from two V8 fragments of purified nonrecombinant eIF-2B epsilon in the deduced amino acid sequence of the cDNA. The open reading frame of the cDNA began with the third nucleotide of the cDNA with the first AUG codon at nucleotide 522. Mutational analysis of pA1 indicated that the cDNA did not code for full-length eIF-2B epsilon. Seven missing codons of the reading-frame and the 71 nucleotide 5' non-coding region of the eIF-2B epsilon mRNA were obtained by 5' RACE. A human eIF-2B epsilon cDNA fragment, which corresponded to a similar 2.3 kb fragment generated by digestion of the rabbit pA1 cDNA with EcoRI, was isolated from a human histiocytic lymphoma (U-937) cell cDNA library constructed in lambda gt10. The nucleotide and amino acid sequences were highly conserved between the rabbit and human cDNAs, showing approx. 90% sequence identity within the open reading frame. Northern and Western blot analyses of reticulocyte lysate and other rabbit tissue extracts indicated that the eIF-2B epsilon polypeptide has a similar apparent molecular weight in all tissues examined, and is coded for by a single approximately 2.8 kilobase mRNA species which is ubiquitously expressed.