The nuclear migration protein NUDF/LIS1 forms a complex with NUDC and BNFA at spindle pole bodies.
Nuclear migration depends on microtubules, the dynein motor complex, and regulatory components like LIS1 and NUDC. We sought to identify new binding partners of the fungal LIS1 homolog NUDF to clarify its function in dynein regulation. We therefore analyzed the association between NUDF and NUDC in Aspergillus nidulans. NUDF and NUDC directly interacted in yeast two-hybrid experiments via NUDF's WD40 domain. NUDC-green fluorescent protein (NUDC-GFP) was localized to immobile dots in the cytoplasm and at the hyphal cortex, some of which were spindle pole bodies (SPBs). We showed by bimolecular fluorescence complementation microscopy that NUDC directly interacted with NUDF at SPBs at different stages of the cell cycle. Applying tandem affinity purification, we isolated the NUDF-associated protein BNFA (for binding to NUDF). BNFA was dispensable for growth and for nuclear migration. GFP-BNFA fusions localized to SPBs at different stages of the cell cycle. This localization depended on NUDF, since the loss of NUDF resulted in the cytoplasmic accumulation of BNFA. BNFA did not bind to NUDC in a yeast two-hybrid assay. These results show that the conserved NUDF and NUDC proteins play a concerted role at SPBs at different stages of the cell cycle and that NUDF recruits additional proteins specifically to the dynein complex at SPBs.
Helmstaedt
Kerstin
K
Molekulare Mikrobiologie und Genetik, Institut für Mikrobiologie und Genetik, Georg-August-Universität Göttingen, Grisebachstrasse 8, D-37077 Göttingen, Germany.
Laubinger
Karen
K
Vosskuhl
Katja
K
Bayram
Ozgür
O
Busch
Silke
S
Hoppert
Michael
M
Valerius
Oliver
O
Seiler
Stephan
S
Braus
Gerhard H
GH
D000595:Amino Acid Sequence; D001233:Aspergillus nidulans / Q000166:cytology* / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D004398:Dyneins / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism; D008941:Spindle Apparatus / Q000378:metabolism; D020798:Two-Hybrid System Techniques
D005656:Fungal Proteins; D009687:Nuclear Proteins; D004398:Dyneins