Abstract
Metabolic labeling of proteins with a stable isotope (15N) in intact Arabidopsis plants was used for accurate determination by mass spectrometry of differences in protein abundance between plasma membranes isolated from leaves and roots. In total, 703 proteins were identified, of which 188 were predicted to be integral membrane proteins. Major classes were transporters, receptors, proteins involved in membrane trafficking and cell wall-related proteins. Forty-one of the integral proteins, including nine of the 13 isoforms of the PIP (plasma membrane intrinsic protein) aquaporin subfamily, could be identified by peptides unique to these proteins, which made it possible to determine their relative abundance in leaf and root tissue. In addition, peptides shared between isoforms gave information on the proportions of these isoforms. A comparison between our data for protein levels and corresponding data for mRNA levels in the widely used database Genevestigator showed an agreement for only about two thirds of the proteins. By contrast, localization data available in the literature for 21 of the 41 proteins show a much better agreement with our data, in particular data based on immunostaining of proteins and GUS-staining of promoter activity. Thus, although mRNA levels may provide a useful approximation for protein levels, detection and quantification of isoform-specific peptides by proteomics should generate the most reliable data for the proteome.
Highlights
The plasma membrane constitutes the outer border of the cell
We have chosen to focus on integral membrane proteins, as predicted by Phobius [1], and compared the protein composition of plasma membranes isolated from leaf and root tissue from eight-week-old Arabidopsis plants grown on a liquid medium
41 of the 188 integral proteins identified by the software Mascot (Table S1) generated reliable 14N/15N peptide spectra and were identified with at least one unique peptide (Table 2, Table S2), whereas the remaining 147 proteins were excluded due to unreliable peptide spectra, or, most commonly, because they were only identified with peptides shared by other proteins
Summary
The plasma membrane constitutes the outer border of the cell. This position implies that properties of the plasma membrane will determine almost all types of communication and other interactions between the cell interior and its surrounding environment. Due to the very diverse functions of different organs and tissues, the plasma membrane is likely to be the most diverse membrane of the cell, with a protein composition that varies according to its location in the plant, as well as to developmental stage and environment. We have chosen to focus on integral membrane proteins, as predicted by Phobius [1], and compared the protein composition of plasma membranes isolated from leaf and root tissue from eight-week-old Arabidopsis plants grown on a liquid medium. At this age, the leaf rosette contains leaves of all developmental stages, from fully expanded to newly developed, and the root system is still growing. The relative abundance of proteins in leaves and roots was determined by metabolic labeling of proteins with a stable isotope (15N) and subsequent proteomic analysis using mass spectrometry (MS)
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