In vivo cross-linking combined with mass spectrometry analysis reveals receptor-like kinases and Ca(2+) signalling proteins as putative interaction partners of pollen plasma membrane H(+) ATPases.
During fertilisation in plants, pollen grains germinate and generate a pollen tube which grows through the style tissue to the egg apparatus delivering the two sperm cells for fertilisation. For this process, adaption to specific environmental conditions and communication between male and female organs are essential, requiring the sensing of internal and external signals which are translated into tube growth. The plasma membrane (PM) H(+) ATPase energises the pollen plasma membrane for nutrient, ion and water uptake, but additionally, its activity directly affects the germination frequency and drives the elongation of pollen tubes. A combination of in vivo cross-linking with para-formaldehyde, immunoaffinity purification of cross-linked PM H(+) ATPase complexes and subsequent mass spectrometry analysis revealed putative interaction partners of the PM H(+) ATPase of lily pollen, which are possibly involved in the perception and transduction of intra- and extracellular signals. Major interactions partners included (i) membrane-localised receptor-like kinases (RLKs) with the leucine-rich repeat RLKs (LRR-RLKs) forming the largest group, (ii) interacting protein kinases, phosphatases, WD-40 domain proteins and 14-3-3 proteins that may transduce intracellular, phosphorylation-dependent signals and (iii) specific cytosolic Ca(2+) signatures may be decoded by interacting Ca(2+) sensor proteins, calmodulin and calmodulin-like proteins, and Ca(2+)-dependent protein kinases, which were all identified as interaction partners of the PM H(+) ATPase in lily pollen. These identified interaction partners suggest new putative regulation mechanisms of the PM H(+) ATPase in general and new insights in regulating pollen tube growth rates in particular. Furthermore, the optimised experimental strategy can be applied to other non-model organisms to identify membrane protein interactions.


CONCLUSIONS
Membrane proteomics is still very challenging due to the low abundance and poor solubility of membrane proteins. Furthermore, membrane protein interaction studies in a non-model organism like Lilium longiflorum require an unbiased preparation and detection approach. The presented strategy to identify putative interaction partners of the PM H(+) ATPase by using a combination of different biochemical techniques, i.e. in vivo crosslinking, immunoaffinity purification and mass spectrometry without the need of genetic engineering, transformation or other molecular biology techniques can be easily transferred to other protein interaction studies. The well characterised interaction of the PM H(+) ATPase with regulating 14-3-3 proteins served as an intrinsic control to proof the suitability and reliability of the presented strategy, whilst newly identified interaction partners may indicate novel regulation mechanisms of the PM H(+) ATPase.
Molecular Plant Biophysics and Biochemistry, Department of Molecular Biology, University of Salzburg, Billrothstr. 11, 5020 Salzburg, Austria; Max-Planck-Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany. Electronic address: H.Pertl@uni-hohenheim.de.
Heidi
H
Pertl-Obermeyer
Max-Planck-Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany.
Waltraud X
WX
Schulze
Molecular Plant Biophysics and Biochemistry, Department of Molecular Biology, University of Salzburg, Billrothstr. 11, 5020 Salzburg, Austria.
Gerhard
G
Obermeyer
D020013:Calcium Signaling; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000737:chemistry*; D005557:Formaldehyde / Q000737:chemistry*; D027762:Lilium / Q000737:chemistry / Q000378:metabolism*; D013058:Mass Spectrometry; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D053205:Pollen Tube / Q000737:chemistry / Q000378:metabolism*; D011108:Polymers / Q000737:chemistry*; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000378:metabolism*; D020794:Receptor Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D003432:Cross-Linking Reagents; D010940:Plant Proteins; D011108:Polymers; D005557:Formaldehyde; D020794:Receptor Protein-Tyrosine Kinases; D006180:Proton-Translocating ATPases; C003043:paraform
In vivo cross-linking; Lilium longiflorum; Membrane proteomics; Pollen; Protein-protein interaction; Signal transduction