Figure 5. Mpp6 and Rrp47 stimulate Rrp6 activity and contribute to Mtr4 recruitment.

(A) Both Mpp6 and Rrp47 stimulate Rrp6 activity in degradation of a single-stranded 5’ fluorescein-labeled 49 nt polyA RNA in Exo11^Dis3/Rrp6. Initial rates from triplicate experiments shown, with error bars representing plus or minus one standard deviation. (B) Mpp6, but not Rrp47, requires the Rrp6 lasso to stimulate Rrp6 activity on 5’ fluorescein-labeled 49 nt polyA RNA. Initial rates from triplicate experiments are shown, with error bars representing plus or minus one standard deviation. (C) Mpp6 contributes to Mtr4 recruitment in Exo11^Dis3/Rrp6. Analytical gel filtration experiments were performed with Exo10^Dis3 and Exo11^Dis3/Rrp6, Rrp47, Mtr4, with various truncations of Mpp6 and Rrp6 as indicated. Bar graphs represent the ratio of Mtr4 (122 kDa) to Dis3 (114 kDa) in peak fractions of the complex (Figure 5—figure supplement 1C), as calculated by densitometric analysis of the fractions on SDS-PAGE, with error bars representing plus or minus one standard deviation.

DOI: http://dx.doi.org/10.7554/eLife.29062.011

Figure 5—figure supplement 1. Superposition of Exo12^{Dis3exo-endo-/Rrp6exo-/Mpp6Min}onto Exo11^{Dis3exo-endo-/Rrp6exo-} (PDB 5K36) and Mtr4 interactions with exosomes as analyzed by gel filtration and SDS-PAGE.

(A) Overall structures with the S1/KH ring in orange (Rrp40) and wheat (Csl4 and Rrp4), the PH-like ring in grey, Dis3 in pink or red, and Rrp6 in teal or cyan. Select subunits are labeled. (B) Close up of the Rrp6 CAT and HRDC domains to illustrate rotations and displacements relative to the two structures. (C) Representative SDS-PAGE of analytical gel filtration runs (Superdex 200 Increase) of the indicated Mtr4-exosome combinations presented in Figure 5C, with each lane representing an independent fraction. Gels were Sypro Ruby stained and the ratio of Mtr4 to Dis3 was calculated via densitometry within lanes indicated by solid lines above each gel.

DOI: http://dx.doi.org/10.7554/eLife.29062.012