Universität zu Köln

Waaijers, Selma, Munoz, Javier ORCID: 0000-0003-3288-3496, Berends, Christian, Ramalho, Joao J., Goerdayal, Soenita S., Low, Teck Y., Zoumaro-Djayoon, Adja D., Hoffmann, Michael, Koorman, Thijs, Tas, Roderick P., Harterink, Martin ORCID: 0000-0002-8256-6651, Seelk, Stefanie, Kerver, Jana, Hoogenraad, Casper C., Bossinger, Olaf, Tursun, Baris ORCID: 0000-0001-7293-8629, van den Heuvel, Sander, Heck, Albert J. R. and Boxem, Mike ORCID: 0000-0003-3966-4173 (2016). A tissue-specific protein purification approach in Caenorhabditis elegans identifies novel interaction partners of DLG-1/Discs large. BMC Biol., 14. LONDON: BIOMED CENTRAL LTD. ISSN 1741-7007

Full text not available from this repository.

Abstract

Background: Affinity purification followed by mass spectrometry (AP/MS) is a widely used approach to identify protein interactions and complexes. In multicellular organisms, the accurate identification of protein complexes by AP/MS is complicated by the potential heterogeneity of complexes in different tissues. Here, we present an in vivo biotinylation-based approach for the tissue-specific purification of protein complexes from Caenorhabditis elegans. Tissue-specific biotinylation is achieved by the expression in select tissues of the bacterial biotin ligase BirA, which biotinylates proteins tagged with the Avi peptide. Results: We generated N- and C-terminal tags combining GFP with the Avi peptide sequence, as well as four BirA driver lines expressing BirA ubiquitously and specifically in the seam and hyp7 epidermal cells, intestine, or neurons. We validated the ability of our approach to identify bona fide protein interactions by identifying the known LGL-1 interaction partners PAR-6 and PKC-3. Purification of the Discs large protein DLG-1 identified several candidate interaction partners, including the AAA-type ATPase ATAD-3 and the uncharacterized protein MAPH-1.1. We have identified the domains that mediate the DLG-1/ATAD-3 interaction, and show that this interaction contributes to C. elegans development. MAPH-1.1 co-purified specifically with DLG-1 purified from neurons, and shared limited homology with the microtubule-associated protein MAP1A, a known neuronal interaction partner of mammalian DLG4/PSD95. A CRISPR/Cas9-engineered GFP::MAPH-1.1 fusion was broadly expressed and co-localized with microtubules. Conclusions: The method we present here is able to purify protein complexes from specific tissues. We uncovered a series of DLG-1 interactors, and conclude that ATAD-3 is a biologically relevant interaction partner of DLG-1. Finally, we conclude that MAPH-1.1 is a microtubule-associated protein of the MAP1 family and a candidate neuron-specific interaction partner of DLG-1.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Waaijers, Selma UNSPECIFIED UNSPECIFIED UNSPECIFIED Munoz, Javier UNSPECIFIED orcid.org/0000-0003-3288-3496 UNSPECIFIED Berends, Christian UNSPECIFIED UNSPECIFIED UNSPECIFIED Ramalho, Joao J. UNSPECIFIED UNSPECIFIED UNSPECIFIED Goerdayal, Soenita S. UNSPECIFIED UNSPECIFIED UNSPECIFIED Low, Teck Y. UNSPECIFIED UNSPECIFIED UNSPECIFIED Zoumaro-Djayoon, Adja D. UNSPECIFIED UNSPECIFIED UNSPECIFIED Hoffmann, Michael UNSPECIFIED UNSPECIFIED UNSPECIFIED Koorman, Thijs UNSPECIFIED UNSPECIFIED UNSPECIFIED Tas, Roderick P. UNSPECIFIED UNSPECIFIED UNSPECIFIED Harterink, Martin UNSPECIFIED orcid.org/0000-0002-8256-6651 UNSPECIFIED Seelk, Stefanie UNSPECIFIED UNSPECIFIED UNSPECIFIED Kerver, Jana UNSPECIFIED UNSPECIFIED UNSPECIFIED Hoogenraad, Casper C. UNSPECIFIED UNSPECIFIED UNSPECIFIED Bossinger, Olaf UNSPECIFIED UNSPECIFIED UNSPECIFIED Tursun, Baris UNSPECIFIED orcid.org/0000-0001-7293-8629 UNSPECIFIED van den Heuvel, Sander UNSPECIFIED UNSPECIFIED UNSPECIFIED Heck, Albert J. R. UNSPECIFIED UNSPECIFIED UNSPECIFIED Boxem, Mike UNSPECIFIED orcid.org/0000-0003-3966-4173 UNSPECIFIED
URN:	urn:nbn:de:hbz:38-266670
DOI:	10.1186/s12915-016-0286-x
Journal or Publication Title:	BMC Biol.
Volume:	14
Date:	2016
Publisher:	BIOMED CENTRAL LTD
Place of Publication:	LONDON
ISSN:	1741-7007
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language MASS SPECTROMETRY DATA; C.-ELEGANS; AFFINITY PURIFICATION; ESCHERICHIA-COLI; MOLECULAR CHARACTERIZATION; SYSTEMATIC IDENTIFICATION; SACCHAROMYCES-CEREVISIAE; MICROTUBULE DYNAMICS; CELL POLARITY; IN-VIVO Multiple languages Biology Multiple languages
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/26667