Self-association of the spindle pole body-related intermediate filament protein Fin1p and its phosphorylation-dependent interaction with 14-3-3 proteins in yeast.
The Fin1 protein of the yeast Saccharomyces cerevisiae forms filaments between the spindle pole bodies of dividing cells. In the two-hybrid system it binds to 14-3-3 proteins, which are highly conserved proteins involved in many cellular processes and which are capable of binding to more than 120 different proteins. Here, we describe the interaction of the Fin1 protein with the 14-3-3 proteins Bmh1p and Bmh2p in more detail. Purified Fin1p interacts with recombinant yeast 14-3-3 proteins. This interaction is strongly reduced after dephosphorylation of Fin1p. Surface plasmon resonance analysis showed that Fin1p has a higher affinity for Bmh2p than for Bmh1p (K(D) 289 versus 585 nm). Sequences in both the central and C-terminal part of Fin1p are required for the interaction with Bmh2p in the two-hybrid system. In yeast strains lacking 14-3-3 proteins Fin1 filament formation was observed, indicating that the 14-3-3 proteins are not required for this process. Fin1 also interacts with itself in the two-hybrid system. For this interaction sequences at the C terminus, containing one of two putative coiled-coil regions, are sufficient. Fin1p-Fin1p interactions were demonstrated in vivo by fluorescent resonance energy transfer between cyan fluorescent protein-labeled Fin1p and yellow fluorescent protein-labeled Fin1p.
van Hemert
Martijn J
MJ
Section Yeast Genetics, Institute of Molecular Plant Sciences, Leiden University, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands.
Deelder
André M
AM
Molenaar
Chris
C
Steensma
H Yde
HY
van Heusden
G Paul H
GP
D048948:14-3-3 Proteins; D001665:Binding Sites; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer; D010449:Peptide Mapping; D010766:Phosphorylation; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000166:cytology; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D008941:Spindle Apparatus / Q000737:chemistry / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism*
D048948:14-3-3 Proteins; C097164:BMH2 protein, S cerevisiae; D003598:Cytoskeletal Proteins; C456282:FIN1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014446:Tyrosine 3-Monooxygenase