Binary protein interactome mapping of the Giardia lamblia proteasome lid reveals extra proteasomal functions of GlRpn11.

Abstract

The assembly of the 26S proteasome, a multi-subunit complex for regulated protein turnover, proceeds via the formation of intermediates. Giardia lamblia does not encode proteasome regulatory subunit Rpn12 or proteasome complex subunit Sem1, two proteins crucial for assembling the proteasome lid. To understand how the interactions between the giardial proteasome lid subunits may have changed to compensate for their absence, we used yeast two-hybrid to generate a binary interactome map of Giardia's lid subunits. Most interactions within the Giardia lid are stronger than Saccharomyces cerevisiae lid, which may compensate for Rpn12 and Sem1 absence. A notable exception was the weaker interaction between the two non-ATPase lid subunits, GlRpn11 and GlRpn8, compared to the strong interaction between yeast orthologs Rpn11 and Rpn8. The Rpn11-Rpn8 dimer provides a platform for lid assembly. Their interaction involves the insertion of a methionine residue of Rpn11 into a hydrophobic pocket of Rpn8. Molecular modeling indicates that GlRpn8's pocket is wider, reconciling the experimental observation of its weak interaction with GlRpn11. This weaker interaction may have evolved to support proteasome-independent functions of GlRpn11, which localizes to multiple subcellular regions, including the mitosomes, where other proteasome subunits cannot be detected. Functional complementation in yeast shows that GlRpn11 can influence mitochondrial function and distribution. Together these observations show that GlRpn11 functions at the mitosome. Thus, this parasite's proteasome lid has a simpler subunit architecture than that of yeast with structural attributes to support dual functionalities for GlRpn11. Such parasite-specific proteasome features provide opportunities for controlling parasite transmission.