Abstract
Petunia inflata small CDPK-interacting protein 1 (PiSCP1) was identified as a pollen expressed PiCDPK1 interacting protein using the yeast two hybrid system and the interaction confirmed using pull-down and phosphorylation assays. PiSCP1 is pollen specific and shares amino acid homology with uncharacterized proteins from diverse species of higher plants, but no protein of known function. Expression of PiSCP1-GFP in vivo inhibited pollen tube growth and was shown to localize to peroxisomes in growing pollen tubes. As PiCDPK1 is plasma membrane localized, we investigated the localization of a second isoform, PiCDPK2, and show that it co-localizes to peroxisomes with PiSCP1 and that the two proteins interact in the yeast 2 hybrid interaction assay, suggesting that interaction with the latter CDPK isoform is likely the one of biological relevance. Both PiCDPK2 and PiSCP1 affect pollen tube growth, presumably by mediating peroxisome function, however how they do so is currently not clear.
Highlights
Ca2+ has long been known to play a key regulatory role in pollen tube growth [1,2,3,4]
Though Petunia inflata small calcium dependent protein kinases (CDPKs)-interacting protein 1 (PiSCP1) was identified as a protein that interacted with PiCDPK1, we have previously shown that PiCDPK1 exhibits plasma membrane localization that is dependent on potential myristoylation and palmitoylation sites at the N-terminus, a second isoform—PiCDPK2, exhibits localization pattern reminiscent of that of PiSCP1 [15]
The data presented in this manuscript provides evidence that a previously unknown pollen specific protein, PiSCP1, interacts with calcium dependent proteins kinases and localizes to peroxisomes
Summary
Ca2+ has long been known to play a key regulatory role in pollen tube growth [1,2,3,4]. As plants appear to employ a combination of strategies to functionally specialize individual CDPKs, including tissue-specific distribution, subcellular localization, and enzyme kinetics and properties [23,24] it is important to recognize that methods for identifying substrates are based on biochemical interaction. With this in mind we sought to identify CDPK substrates in pollen tubes by screening a pollen cDNA yeast 2 hybrid library using the kinase domain of PiCDPK1 as bait and report the identification and characterization of Petunia inflata Small CDPK interacting Protein 1. (PiSCP1), and demonstrate that its protein product co-localizes to peroxisomes with PiCDPK2
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