Spt16 and Pob3 of Saccharomyces cerevisiae form an essential, abundant heterodimer that is nuclear, chromatin-associated, and copurifies with DNA polymerase alpha.

Previously we showed that the yeast proteins Spt16 (Cdc68) and Pob3 are physically associated, and interact physically and genetically with the catalytic subunit of DNA polymerase alpha, Pol1 [Wittmeyer and Formosa (1997) Mol. Cell. Biol. 17, 4178-4190]. Here we show that purified Spt16 and Pob3 form a stable, abundant, elongated heterodimer and provide evidence that this is the functional form of these proteins. Genetic interactions between mutations in SPT16 and POB3 support the importance of the Spt16-Pob3 interaction in vivo. Spt16, Pob3, and Pol1 proteins were all found to localize to the nucleus in S. cerevisiae. A portion of the total cellular Spt16-Pob3 was found to be chromatin-associated, consistent with the proposed roles in modulating chromatin function. Some of the Spt16-Pob3 complex was found to copurify with the yeast DNA polymerase alpha/primase complex, further supporting a connection between Spt16-Pob3 and DNA replication.     

Identification of developmentally expressed proteins that functionally interact with Nedd4 ubiquitin ligase.

Nedd4 is a HECT domain-containing ubiquitin ligase that mediates ubiquitylation and proteasome degradation of target proteins. The molecular basis for the interaction of Nedd4 with substrates lies in its WW domains, which can bind proline-rich (PY) domains in target proteins. Nedd4 is a developmentally expressed protein and may have a fundamental role to play in embryonic processes. However, whether Nedd4 has such a function is currently unknown, in part because few developmentally regulated ubiquitylation substrates have been identified or characterized. We have carried out a yeast two-hybrid screen and identified four proteins expressed in the mid-gestation embryo that are able to interact with Nedd4. Characterization of their functional interaction with Nedd4 in vitro and in vivo demonstrated that three of the four are bona fide Nedd4 binding partners, and two have the capacity to be ubiquitylation substrates. One of these is the first identified nonviral substrate for Nedd4-mediated monoubiquitylation. Interestingly, neither of these two ubiquitylated proteins interacts with Nedd4 through PY-mediated mechanisms. For one of the three Nedd4 binding partners, there was no discernable evidence of ubiquitylation. However, this protein clearly associates with Nedd4 through its PY domains and can alter the location of Nedd4 in cells, suggesting a role other than as a ubiquitylation substrate.