A Proteomic Approach to Identification of Transmembrane Proteins and Membrane-anchored Proteins of Arabidopsis thaliana by Peptide Sequencing

Abstract

A proteomic approach was developed for the identification of membrane-bound proteins of Arabidopsis thaliana. A subcellular fraction enriched in vacuolar membranes was prepared from 4-week-old plants and was washed with various agents to remove peripheral membrane proteins and contaminating soluble proteins. The remaining membrane-bound proteins were then subjected to proteomic analysis. Given that these proteins were resolved poorly by standard two-dimensional gel electrophoresis, we subjected them instead to SDS-polyacrylamide gel electrophoresis and to protein digestion within gel slices with lysylendopeptidase. The resulting peptides were separated by reverse-phase high-performance liquid chromatography and subjected to Edman sequencing. From the 163 peptide peaks analyzed, 69 peptide sequences were obtained, 64 of which were informative. The proteins corresponding to these peptide sequences were identified as belonging to 42 families, including two subfamilies, by comparison with the protein sequences predicted from annotation of the A. thaliana genome. A total of 34 proteins was identified definitively with protein-specific peptide sequences. Transmembrane proteins detected in the membrane fraction included transporters, channels, receptors, and unknown molecules, whereas the remaining proteins, categorized as membrane-anchored proteins, included small GTPases, GTPase binding proteins, heat shock protein 70-like proteins, ribosomal proteins, and unknown proteins. These membrane-anchored proteins are likely attached to membranes by hydrophobic anchor molecules or through tight association with other membrane-bound proteins. This proteomic approach has thus proved effective for the identification of membrane-bound proteins.

Genome-Wide Annotation of Remorins, a Plant-Specific Protein Family: Evolutionary and Functional Perspectives

“…Group 1b contains the first discovered remorins, StREM1.3 (Reymond et al, 1996) and LeREM1 (Bariola et al, 2004; now named SlREM1.2), to which most of the current literature refers. The described Arabidopsis remorin AtDBP (AtREM1.3) and most remorins currently identified in proteomic screens of membrane preparations also cluster in this group (Watson et al, 2003;Marmagne et al, 2004;Mongrand et al, 2004;Sazuka et al, 2004;Nelson et al, 2006;Valot et al, 2006). Association with membranes thus seems to be a common feature of group 1b remorins.…”

“…Several proteomic approaches on PM preparations from tobacco (Nicotiana tabacum) leaves and Arabidopsis (Arabidopsis thaliana) seedlings now suggest localization of remorins in association with PMs (Watson et al, 2003;Marmagne et al, 2004;Mongrand et al, 2004;Sazuka et al, 2004;Nelson et al, 2006;Valot et al, 2006). Interestingly, remorins have also been found in detergent-resistant membrane fractions, called lipid rafts (Mongrand et al, 2004;Shahollari et al, 2004;Bhat and Panstruga, 2005;Laloi et al, 2007;Lefebvre et al, 2007).…”

Identification of a Vacuolar Sucrose Transporter in Barley and Arabidopsis Mesophyll Cells by a Tonoplast Proteomic Approach

“…Activities of marker enzymes in the vacuolar preparations of barley mesophyll protoplasts Marker enzyme activities for mitochondria (NAD-malic dehydrogenase), chloroplasts (NADP-gyceraldehydphosphate dehydrogenase, Glc-phosphate isomerase), ER (NADPH-cytochrome-c-reductase), and cytosol (Glc-phosphate isomerase, NAD-malic dehydrogenase) were measured in protoplast and vacuolar fractions. The percentage of contamination was calculated assuming an exclusive localization of a-Mannosidase in the vacuole (Martinoia et al, 1981 Carter et al (2004;a), Shimaoka et al (2004;, Szponarski et al (2004;, and Sazuka et al (2004; (Nakamura et al, 2006). Moreover, there were also nine proteins with at least one transmembrane helix for which a function has yet not been elucidated (Table II).…”

“…For example, in Arabidopsis, vacuoles accumulate mainly monosaccharides, whereas in barley, fructans may be synthesized and stored in vacuoles. To identify proteins that are particular for the vacuolar function in barley, we compared the vacuolar proteins found in barley to proteins identified in the four Arabidopsis vacuolar proteomic approaches (Carter et al, 2004;Sazuka et al, 2004;Shimaoka et al, 2004;Szponarski et al, 2004). We searched the 88 identified vacuolar proteins for the closest homologs in the Arabidopsis genome.…”

“…Thus far, vacuolar proteomic approaches have been described using Arabidopsis (Arabidopsis thaliana) vacuoles (Carter et al, 2004;Sazuka et al, 2004;Shimaoka et al, 2004;Szponarski et al, 2004). Currently, no data are available for monocotyledonous cereal plants.…”