Phosphoproteomics of the Arabidopsis Plasma Membrane and a New Phosphorylation Site Database[W]

Abstract

Functional genomic technologies are generating vast amounts of data describing the presence of transcripts or proteins in plant cells. Together with classical genetics, these approaches broaden our understanding of the gene products required for specific responses. Looking to the future, the focus of research must shift to the dynamic aspects of biology: molecular mechanisms of function and regulation. Phosphorylation is a key regulatory factor in all aspects of plant biology; but it is difficult, if not impossible, for most researchers to identify in vivo phosphorylation sites within their proteins of interest. We have developed a large-scale strategy for the isolation of phosphopeptides and identification by mass spectrometry (Nü hse et al., 2003b). Here, we describe the identification of more than 300 phosphorylation sites from Arabidopsis thaliana plasma membrane proteins. These data will be a valuable resource for many fields of plant biology and overcome a major impediment to the elucidation of signal transduction pathways. We present an analysis of the characteristics of phosphorylation sites, their conservation among orthologs and paralogs, and the existence of putative motifs surrounding the sites. These analyses yield general principles for predicting other phosphorylation sites in plants and provide indications of specificity determinants for responsible kinases. In addition, more than 50 sites were mapped on receptor-like kinases and revealed an unexpected complexity of regulation. Finally, the data also provide empirical evidence on the topology of transmembrane proteins. This information indicates that prediction programs incorrectly identified the cytosolic portion of the protein in 25% of the transmembrane proteins found in this study. All data are deposited in a new searchable database for plant phosphorylation sites maintained by PlantsP (http://plantsp.sdsc.edu) that will be updated as the project expands to encompass additional tissues and organelles.

ArabidopsisPEN3/PDR8, an ATP Binding Cassette Transporter, Contributes to Nonhost Resistance to Inappropriate Pathogens That Enter by Direct Penetration

“…ABC transporters have been shown to require phosphorylation for activity or regulation (Kolling, 2002). A recent study of phosphorylated plasma membrane proteins identified PEN3 as a target of flagellin-induced phosphorylation (Nü hse et al, 2004), supporting the idea that PEN3 functions in nonhost resistance and defenses elicited by nonspecific elicitors such as flagellin. Consistent with this idea, PEN3 expression was 2.5-fold higher in flg22 peptide-treated seedlings compared with control seedlings, and this induction was dependent on the FLS2 receptor-like kinase (see Supplemental Table 2 online) (Gomez-Gomez and Boller, 2000).…”

“…Recently, proteomic studies of different organelles have placed PEN3 in the plasma membrane, mitochondria, and chloroplasts (Brugiere et al, 2004;Kleffmann et al, 2004;Nü hse et al, 2004). Given its high abundance, PEN3 is likely a common contaminant in organelle preparations and unlikely to be localized in all three subcellular structures.…”

A phospholipid uptake system in the model plant Arabidopsis thaliana

“…In this context, ALA10 could also function as a sensor of phospholipids in the soil environment that initiates signalling events leading to secretion of phosphatases. Like transceptors 32,33 , ALA10 is subject to phosphorylation in planta at serine and threonine residues 20,21 , which suggests that it is subject to tight regulation. One of the first signs of phosphate starvation in A. thaliana is that the root enters a determinate developmental programme in which primary root growth is arrested and cell differentiation is promoted, thereby stimulating lateral root growth and increasing the root surface area 34 .…”

“…Large-scale proteomic studies suggest that ALA10 exists as a phosphoprotein at the plasma membrane 20,21 . In animal cells, P4-ATPases at this location constantly remove PS from the outer plasma membrane leaflet, which protects cells, as macrophages engulf cells with this phospholipid exposed at their surface 22 .…”

“…However, the localization pattern of the newly identified Class 7 P4-ATPases was less clear. ALA10 has been identified in largescale phosphoproteomics studies of the plasma membrane 20,21 , ALA6 has been localized to the plasma membrane but also to highly mobile endomembrane structures 18 , whereas ALA3, in an outgroup of its own, has been localized to the Golgi apparatus 15 . This prompted us to investigate the localization of ALA10 in more detail.…”