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BMC Biol. 2016 Aug 9;14:66. doi: 10.1186/s12915-016-0286-x.

A tissue-specific protein purification approach in Caenorhabditis elegans identifies novel interaction partners of DLG-1/Discs large.

Author information

1
Developmental Biology, Department of Biology, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands.
2
Present address: Department of Physiology, Radboud University Medical Center, Geert Grooteplein 26, 6525 GA, Nijmegen, The Netherlands.
3
Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands.
4
Netherlands Proteomics Centre, Padualaan 8, 3584 CH, Utrecht, The Netherlands.
5
Present address: Proteomics Unit, Spanish National Cancer Research Centre (CNIO), ProteoRed-ISCIII, 28029, Madrid, Spain.
6
Institut für Wissenschaftliche Medizin, D-40591, Düsseldorf, Germany.
7
Present address: Center for Cancer Research and Department of Pathology, Massachusetts General Hospital and Harvard Medical School Department of Pathology, 149 13th Street, 02129, Charlestown, MA, USA.
8
Cell Biology, Department of Biology, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands.
9
Berlin Institute for Medical Systems Biology (BIMSB), Max Delbrueck Center for Molecular Medicine (MDC) in the Helmholtz Association, Robert-Roessle-Strasse 10, Berlin, 13125, Germany.
10
Molecular Cell Biology, Anatomy I, University of Cologne, D-50937, Cologne, Germany.
11
Berlin Institute for Medical Systems Biology (BIMSB), Max Delbrueck Center for Molecular Medicine (MDC) in the Helmholtz Association, Robert-Roessle-Strasse 10, Berlin, 13125, Germany. baris.tursun@mdc-berlin.de.
12
Developmental Biology, Department of Biology, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands. m.boxem@uu.nl.

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.

KEYWORDS:

Affinity purification; C. elegans; Discs large; Mass spectrometry; Microtubule-associated; Protein complex; Tissue-specific

PMID:
27506200
PMCID:
PMC4977824
DOI:
10.1186/s12915-016-0286-x
[Indexed for MEDLINE]
Free PMC Article

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