Abstract
Eukaryotic cells contain an unusually large cytoplasmic pool of P1/P2 phosphoproteins, which form the highly flexible 60S subunit stalk that is required to interact with and activate soluble translation factors. In cells, cytoplasmic P1/P2 proteins are exchanged for ribosome-bound proteins in a process that can modulate ribosome function and translation. Here, we analysed different S. cerevisiae stalk mutants grown under stress conditions that result in eIF2α phosphorylation. These mutants either lack a cytoplasmic pool of stalk proteins or contain free but not ribosome-bound proteins. Only cells that contain free P1/P2 proteins induce eIF2 phosphorylation in vivo in response to glucose starvation or osmotic stress. Moreover, we show that free S. cerevisiae P1/P2 proteins can induce in vitro phosphorylation of the initiation factor eIF2 by stimulating the autophosphorylation and activation of GCN2 kinase. Indeed, these ribosomal proteins do not stimulate other eIF2α kinases, such as PKR and HRI. P1/P2 and the known GCN2 activator deacylated tRNA compete for stimulating the eIF2α kinase activity of GCN2, although the P1/P2 proteins are considerably more active. These findings reveal a capacity of free cytoplasmic ribosomal stalk components to stimulate eIF2α phosphorylation, which in turn would modulate translation in response to specific forms of stress that may be linked with the previously described regulatory function of the ribosomal stalk.
Highlights
The ribosomal stalk is a lateral protuberance of the large ribosomal subunit, which is essential for ribosome function in organisms of all biological Kingdoms
Dramatic differences were observed in both the exponential and the stationary phases of growth: while the level of eIF2a phosphorylation was markedly increased in wild-type cells during the stationary phase, it was very low in the exponential phase and only a modest increase was observed in D4567 cells in the A600 = 2.0 point (Figure 1)
As the cytoplasmic pool of P1/P2 proteins is large and experimental evidence indicates that the free stalk proteins participate in the intracellular stimulation of some ribosome-inactivating proteins (RIP) [16], an additional extraribosomal function of these proteins may not be totally unexpected [46]
Summary
The ribosomal stalk is a lateral protuberance of the large ribosomal subunit, which is essential for ribosome function in organisms of all biological Kingdoms. The stalk is formed by a set of 12 kDa acidic protein dimers that bind to a larger core protein, which in turn interacts with the highly conserved GTPaseassociated region (GAR) of the large rRNA that binds the entire complex to the ribosome (see [1] for a review). The 32 kDa core protein P0 binds to two heterodimers of the 12 kDa P1 and P2 proteins, forming the stalk P0-(P1/P2) complex [2]. The acidic 12 kDa proteins of the yeast eukaryotic stalk are not essential for stalk function but rather, they modulate ribosomal activity [11]
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