Two-hybrid Mpp10p interaction-defective Imp4 proteins are not interaction defective in vivo but do confer specific pre-rRNA processing defects in Saccharomyces cerevisiae

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Oxford University Press

RESUMO

The SSU processome is a large, evolutionarily conserved ribonucleoprotein (RNP), consisting of the U3 snoRNA and at least 28 protein components, that is required for biogenesis of the 18S rRNA. We tested the function of one protein–protein interaction in the SSU processome, Mpp10p–Imp4p, in ribosome biogenesis. Exploiting the reverse two-hybrid system, we screened for mutated Imp4 proteins that were conditionally defective for interaction with Mpp10p. Three different imp4 sequences were isolated that: (i) conferred conditional growth in the two-hybrid strain; (ii) complemented the disrupted imp4; (iii) conferred conditional growth in the context of their normal cellular function; and (iv) resulted in defective pre-rRNA processing at the non-permissive temperatures. Domain swapping revealed that mutations that conferred cold sensitivity resided in the N-terminal coiled-coil domain while mutations in the C-terminus conferred temperature sensitivity. Surprisingly, the mutated Imp4 proteins were not measurably defective for interaction with Mpp10p in the context of the SSU processome. This suggests that other members of the complex may contribute to maintaining the Mpp10p–Imp4p interaction in this large RNP. Since protein–protein interactions are critical for many different aspects of cellular metabolism, our work has implications for the study of other large protein complexes.

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