Project description:SDS22 forms an inactive complex with nascent protein phosphatase-1 (PP1) and Inhibitor-3 (I3). SDS22:PP1:I3 is a substrate for the ATPase p97/VCP, which liberates PP1 for binding to canonical regulatory subunits. The exact role of SDS22 in PP1-holoenzyme assembly remains elusive. Here, we show that SDS22 prevents the aggregation of nascent PP1. In the absence of SDS22, PP1 was gradually lost, resulting in substrate hyperphosphorylation and a proliferation arrest. A human patient with an unstable SDS22 mutant also expressed reduced levels of PP1 and suffered from neurodevelopmental retardation. We furthermore found that SDS22 directly binds to I3 and that this is essential for the stable assembly of SDS22:PP1:I3, the recruitment of p97/VCP, and the extraction of SDS22 during holoenzyme assembly. SDS22 with a disabled I3-binding site co-transfered with PP1 to canonical regulatory subunits, thereby forming non-functional holoenzymes. Our data show that SDS22, by its ability to bind to both PP1 and I3, integrates the major steps of PP1 holoenzyme assembly.
Project description:The goal of the project was to check the differential effect on transcription rate of a sudden degron-induced depletion of Hpr1 protein and its comparison towards the effect of a constitutive absence of Hpr1 protein in a deletion strain. Cells were grown in YP-Raffinose + 0.1 mM CuSO4 at 26ºC to 0.5 OD600. At that cells were spun-down and changed to YP-Galactose without CuSO4 for 30 min at 26ºC. Then cells were shifted to 37ºC to induce degron-mediated proteolysis and recovered after 30 min. Samples include wt, hpr1delta and hpr1-degron after 0 or 30 min growth at 30ºC.
