p23H implicated as cis/trans regulators of AlaXp directed editing for mammalian cell homeostasis

doi:10.1073/pnas.1019400108

The toxicity of mistranslation of serine for alanine appears  to be universal and is prevented in part by the editing activities of alanyl-tRNA synthetases (AlaRSs), which remove serine from mischarged tRNA Ala. The problem of serine mistranslation is so acute that free standing, genome- encoded fragments of the editing domain of AlaRSs are found throughout evolution. These AlaXps are thought to provide functional redundancy of editing. Indeed, archaeal versions rescue the conditional lethality of bacterial cells harboring an editing inactive AlaRs. In mammals, AlaXps are encoded by a gene that fuses coding sequences of a homology of the HSP90 cochaperone p23 (p23 H) to those of AlaXp, to create p23H AlaXp. Not known is whether this fusion protein or various potential splice variants are expressed as editing proficient proteins in mammalian cells. Here we show that both p23H AlaXp and AlaXp alternative splice variants can be detected as proteins in mammalian cells. The variant that ablated p23H and encoded just AlaXp was active in vitro. In contrast, neither the p23H AlaXp fusion protein, nor the mixture of free p23H with AlaXp, was active. Further experiments in a mammalian cell based system showed that RNAi directed suppression of sequences encoding AlaXp led to a serine sensitive increase in the accumulation of misfolded proteins. The results demonstrate the dependence of mammalian cell homeostasis on AlaXp and implicate p23H as a cis and trans acting regulator of its activity.