Study of the interaction of ubiquitin-protein ligase Rsp5p with actin cytoskeleton proteins Bee1p and Lsb1p in S. cerevisiae.
Marta Stawiecka (1), Teresa Zoladek (1)
(1) Department of Genetics, IBB PAS, Warsaw, Poland
Rsp5p ubiquitin ligase plays an important role in endocytosis. It participates in ubiquitination of cargo proteins and affects components of the endocytic machinery. Various data indicate that the endocytic machinery includes some components of the actin cytoskeleton. Bee1p localizes in actin patches and affects both endocytosis and actin polymerization. We have studied the ubiquitination of Bee1p and have found that it is not ubiquitinated. The interaction between Bee1p and Rsp5p was studied using the two hybrid system and no interaction between these two proteins was detected. Lsb1p was chosen as a potential candidate for a mediator in Rsp5p-Bee1p interaction because it interacts with Bee1p and is ubiquitinated. We have demonstrated that Rsp5p affected the level of Lsb1p in the cell, similarly as the level of Bee1p. The level of Lsb1p and Bee1p was lower when an additional copy of RSP5 was present and it was higher in rsp5 mutant as compared to the wild type. Additionally, we tested the potential influence of Rsp5p on the transcription of BEE1 and LSB1. Northern blot analysis showed that the steady state level of LSB1 and BEE1 transcripts was not changed in rsp5 mutant as compared to the wild type strain. These results indicate possible involvement of Rsp5p in Lsb1p and Bee1p synthesis or degradation.