Department of
Protein Science
Project Proposal
Title
Resolving the function of a novel developmental protein (Pifo) by
quantitative mass spectrometric analysis (SILAC) of native protein
complex composition.
Acronym PIFO
PI/Applicant
Heiko Lickert (ISF)
Involved people/institutes/planned authorships
Doris Kinzl (ISF, first author), Karsten Boldt (PROT, shared first author), Marius
Ueffing (PROT, Co-author), Heiko Lickert (ISF, last author, corresponding
author)
Funding/Research programs (Topic: contribution to program/milestone)
POFII, SAM (Topic 2, WP II.1.3.)
Project summary
Pitchfork (Pifo) is a highly conserved functionally non-annotated gene expressed in
mono-ciliated pit cells of the mouse node in early embryonic development. Deletion of
this gene is embryonic lethal with severe heart defects and molecular image analysis
suggests a function in LF-axis formation due to defects in node cilia formation.
In order to investigate the yet unknown function on the protein level, Pifo will be tagged
with SF-TAP (Gloeckner et al., 2007) and transiently expressed in appropriate cellular
systems. Double affinity purification of Pifo-containing native protein complexes and
mass spectrometric complex partner identification will enable to identify cellular
interactors of Pifo.
Interestingly, a potential life threatening mutation in Pifo has been identified in a patient
with laterality defects (DORV) and a sporadic neonatal lethal case with situs inversus. In
order to identify the impact of this mutation in Pifo (PifoR80K) on native complex
formation, quantitative labelling techniques (SILAC) will be implemented and complex
compositions of PifoR80K will be compared to Pifowt protein complexes.
Benefit
Generation of novel functional data for a disease gene and of an entry point to
understand primary cilia signalling. Establishment of quantitative SILAC interaction
proteomics provides this technology for studying other mutation-induced impacts on the
functional protein level.
Effort (Resources/Manpower/ Machine Time for Core Facility)
Cloning and transient expression of Pifowt and PifoR80K (PROT, ISF), setup of SILAC
labelling (PROT), SF-TAP pull-downs (PROT), LC-MSMS (approx. 80 runs, 15 days
machine time, PROT), quantitative analyses (100 hours, PROT). Establishment of cellular
model for cilia assembly and disassembly (PROT, ISF). Validation of potential interactors
(PROT, ISF).
Department of
Protein Science
Project Proposal
Signatures