Modification-Specific Proteomics of Plasma Membrane Proteins:  Identification and Characterization of Glycosylphosphatidylinositol-Anchored Proteins Released upon Phospholipase D Treatment

Abstract

Plasma membrane proteins are displayed through diverse mechanisms, including anchoring in the extracellular leaflet via glycosylphosphatidylinositol (GPI) molecules. GPI-anchored membrane proteins (GPI-APs) are a functionally and structurally diverse protein family, and their importance is well-recognized as they are candidate cell surface biomarker molecules with potential diagnostic and therapeutic applications in molecular medicine. GPI-APs have also attracted interest in plant biotechnology because of their role in root development and cell remodeling. Using a shave-and-conquer concept, we demonstrate that phospholipase D (PLD) treatment of human and plant plasma membrane fractions leads to the release of GPI-anchored proteins that were identified and characterized by capillary liquid chromatography and tandem mass spectrometry. In contrast to phospholipase C, the PLD enzyme is not affected by structural heterogeneity of the GPI moiety, making PLD a generally useful reagent for proteomic investigations of GPI-anchored proteins in a variety of cells, tissues, and organisms. A total of 11 human GPI-APs and 35 Arabidopsis thaliana GPI-APs were identified, representing a significant addition to the number of experimentally detected GPI-APs in both species. Computational GPI-AP sequence analysis tools were investigated for the characterization of the identified GPI-APs, and these demonstrated that there is some discrepancy in their efficiency in classification of GPI-APs and the exact assignment of omega-sites. This study highlights the efficiency of an integrative proteomics approach that combines experimental and computational methods to provide the selectivity, specificity, and sensitivity required for characterization of post-translationally modified membrane proteins.

Disruption of Glycosylphosphatidylinositol-Anchored Lipid Transfer Protein Gene Altered Cuticular Lipid Composition, Increased Plastoglobules, and Enhanced Susceptibility to Infection by the Fungal Pathogen Alternaria brassicicola

“…Asterisks denote statistical differences with respect to the wild-type (* P , 0.05, ** P , 0.01). proteins (Elortza et al, 2006). The results in Figure 3 confirm that LTPG1 is among the GPI proteins anchored to the plasma membrane.…”

“…During posttranslational modification of GPI-anchored proteins in the lumen of the ER, a transamidase recognizes and cleaves the C-terminal hydrophobic region at the omega site. An amide linkage between the ethanolamine of the GPI anchor and the newly generated carboxyl group at the end of the cleaved protein precursor is then created (Udenfriend and Kodukula, 1995;Elortza et al, 2006). As shown in Figure 1, B and C, the LTPG1 protein harbors common structural features of the GPI-anchored proteins, which include the following: (1) an N-terminal secretion domain (amino acid residues 1-22); (2) a hydrophilic spacer region (amino acid residues 162-167); and (3) hydrophobic residues (168-191) at the C terminal end.…”

Arabidopsis LTPG Is a Glycosylphosphatidylinositol-Anchored Lipid Transfer Protein Required for Export of Lipids to the Plant Surface

“…Since some mutants with altered wax load and/or composition do not display this phenotype, quantitative screening using gas chromatography with flame ionization detection (GC-FID) was also performed. With the exception of At1g27950 (ltpg-1, SALK_072495), a gene experimentally identified as a GPI-anchored protein in global proteomics studies Elortza et al, 2006), none of the other mutant lines displayed reductions in stem cuticular wax load (see Supplemental Table 1 online). Therefore, we named this gene LTP-GPI-ANCHORED or LTPG.…”

A Fasciclin-Like Arabinogalactan Protein, GhFLA1, Is Involved in Fiber Initiation and Elongation of Cotton

“…The N-terminal secretion signal sequence and hydrophobic C terminus of AGPs determine where the AGPs are localized in the cell (Showalter, 2001;Ellis et al, 2010). The hydrophobic C terminus of most AtAGPs can be posttranslationally modified by adding a glycosylphosphatidylinositol (GPI) membrane anchor that can be cleaved by phosphatidylinositol-specific phospholipase C and D (Borner et al, 2003;Elortza et al, 2006). GPI-anchored proteins are targeted to the plant cell surface and are likely to be involved in extracellular matrix remodeling and signaling (Borner et al, 2003;Lalanne et al, 2004;Gillmor et al, 2005).…”

“…GPI-anchored proteins are targeted to the plant cell surface and are likely to be involved in extracellular matrix remodeling and signaling (Borner et al, 2003;Lalanne et al, 2004;Gillmor et al, 2005). In Arabidopsis, 14 FLAs are identified as GPI-anchored proteins, eight of which are targets of phosphatidylinositol-specific phospholipase C and D (Borner et al, 2003;Elortza et al, 2003Elortza et al, , 2006.…”