In vivo formation of a stable pentameric (P2α/P1β)-P0-(P1α/P2β) ribosomal stalk complex in Saccharomyces cerevisiae

Heterodimers of acidic proteins P1 alpha/P2 beta and P1 beta/P2 alpha bind to P0 and are fundamental for the assembly of the ribosomal stalk. However, different inconsistencies are found in the literature regarding additional P protein heterodimer formations and their individual interactions with P0. Using the two-hybrid approach, we have found results that help to clarify these interactions. Thus, we have found that neither P1 nor P2 directly interact with P0 unless the endogenous heterodimer partner is being expressed in the cell. In addition, a P2-free amino end is a requisite in these heterodimers for binding to P0. With regard to the two-hybrid interactions between P1 and P2, the known canonical P1 alpha-P2 beta and P1 beta-P2 alpha interactions do not depend on either a free amino end or the presence of endogenous P0, P1 or P2 proteins. Furthermore, the non-canonical P1 beta-P2 beta pair also behaves similarly, although this interaction is weaker. Interestingly, P1 alpha-P2 alpha, P1 alpha-P1 beta and P2 alpha-P2 beta two-hybrid interactions were also detected, although in these cases the endogenous P proteins were involved. Thus, these positive interactions are the consequence of the interaction between two canonical heterodimers. As the ribosome anchorage protein P0 is also necessary, the results suggest that, in vivo, all five P proteins form a complex, independent of the ribosome, containing the two canonical heterodimers and P0. This complex has been isolated in cells expressing a P0 protein unable to bind to the ribosome. Copyright (C) 2010 John Wiley & Sons, Ltd.