Caracterització bioquímica i funcional de ROT1, una proteïna essencial de S.cerevisiae

by Juanes Ortiz, Mª Angeles

Abstract (Summary)
ABSTRACT ROT1 is an essential gene whose inactivation causes defects in cell cycle progression and morphogenesis. Rot1 affects actin cytoskeleton during the cell cycle at two levels. Firstly, it is required for the maintenance of apical growth during bud growth. Secondly, Rot1 is necessary to polarize actin cytoskeleton to the neck region at the end of mitosis; as a consequence of this defect, rot1 cells do not properly form a septum to complete cell division. The inability to polarize actin cytoskeleton at the end of mitosis is not due to a defect in the recruitment of the polarisome scaffold protein Spa2 or the actin cytoskeleton regulators Cdc42 and Cdc24 in the neck region. The previous results point to a connection between Rot1 and the Clb2 cyclin. In fact, an overexpression of CLB2 is toxic when ROT1 is partially inactivated and, reciprocally, a deletion of clb2 suppresses the lethality of the rot1 mutation, which indicates a functional antagonism between Clb2 and Rot1. Several genetic interactions suggest a link between Rot1 and the ubiquitin-proteasome system and in fact, we showed that the Clb2 cyclin is not properly degraded in rot1 mutant cells. Rot1 is primarily located at the endoplasmic reticulum-nuclear membrane facing the lumen. Rot1 migrates more slowly than expected which might suggest post-translational modification. Our results indicate that Rot1 is a protein that is neither GPI-anchored nor O-glycosylated. In contrast, it is N-glycosylated. By a directed mutagenesis of several Asn residues, we identified that the protein is simultaneously glycosylated at N103, N107 and N139. Sequence analysis predicts a transmembrane domain at the C-terminus. This fragment neither affects the targeting of the Rot1 protein to the ER nor its N-glycosylation, although it is important for the anchoring of the protein to the membrane and for its functionality. The existence of a signal sequence at the N-terminus has been suggested. However, deletion of this fragment neither impedes translocation to the ER nor N-glycosylation, but it is required for cell viability. Finally, we found that Rot1 is translocated to the ER by an SRP-independent post-translational mechanism which depends on Sec62.
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Bibliographical Information:

Advisor:Igual García, Juan Carlos; Bañó Aracil, Mª Carmen

School:Universitat de València

School Location:Spain

Source Type:Master's Thesis

Keywords:bioquímica i biologia molecular


Date of Publication:02/20/2008

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