Proteomic analysis of the endoplasmic reticulum from developing and germinating seed of castor ( Ricinus communis )

Abstract

Endoplasmic reticulum (ER) has been prepared and analysed from germinating and developing castor bean endosperm. A combination of one- and two-dimensional (1-D and 2-D) gel electrophoresis was used to study the complexity of sample and protein differences between the two stages. The ER of the developing oilseed is central to the synthesis, sorting and storage of protein and lipid reserves while the germinating seed is concerned with their degradation. Sample complexity has been reduced by separation of ER proteins into lumenal, peripheral membrane and integral membrane subfractions. Membrane proteins pose specific problems in aggregation and binding to passive surfaces. We have overcome this by collection of membranes at density gradient interfaces and by silanization of plastic ware. Several major components have been identified from 1-D gels by N-terminal sequencing and matrix-assisted laser desorption/ionization (MALDI) peptide mass fingerprints. These include protein disulphide isomerase (PDI), calreticulin and developing-ER-specific oleate-12-hydroxylase involved in the biosynthesis of ricinoleic acid. In excess of 300 spots are detectable in each developmental fraction by high sensitivity 2-D gels. This is the first 2-D electrophoretic analysis of plant ER. These gels reveal significant differences between germinating and developing ER. Preparative loading 2-D gels of germinating ER have been run and 14 selected spots characterized by quadrupole time of flight tandem mass spectrometry (Q-TOF MS/MS). Ten of these proteins were assigned function on the basis of identity with existing castor database entries, or by homology with other species. Two proteins, aspartate proteinase precursor and N-carbamyl-L-aminohydrolase-like protein, appear to be absent from developing profiles. Most of the proteins identified are concerned with roles in protein processing and storage, and lipid metabolism which occur in the ER. Data from three of the assigned spots included unidentified peptides indicating the presence of more than one protein in these spots following 2-D electrophoresis. More extensive analysis will have to await developments in genomics but the basic separation technologies to simplify sample identity for a plant ER preparation have been established.

Proteomics of the Chloroplast Envelope Membranes from Arabidopsis thaliana

“…10). Plant mitochondria (11,12), chloroplast (13), plasma membrane (14), peroxisome (15), endoplasmic reticulum (16), and the cell wall (17) have recently been studied with proteomic approaches. Subproteome sample complexity can also be reduced for a more accurate protein location.…”

Proteomics of Rac GTPase Signaling Reveals Its Predominant Role in Elicitor-Induced Defense Response of Cultured Rice Cells

“…So far, the most successful studies are those of subcellular compartments, since they contain a limited number of proteins. Such studies include the proteomics of the chloroplast (Peltier et al, 2000;Friso et al, 2004;Kleffmann et al, 2004;Lonosky et al, 2004;Rose et al, 2004), mitochondria (Kruft et al, 2001;Millar et al, 2001;Bardel et al, 2002), endoplasmic reticulum (Maltman et al, 2002), peroxisome (Fukao et al, 2002), and vacuole (Carter et al, 2004). Proteomic studies of specific stages in development or physiological conditions have been also performed with various plants.…”

High‐value oils from plants

“…With the recent completion of a fourfold coverage of the castor genome, which is publicly available at the TIGR website (http:// castorbean.tigr.org/), and an in-depth analysis of seedspecific ESTs derived from full-length cDNAs (Lu et al, 2007), there is now an unprecedented level of detail available regarding the genes involved in production of one of the most highly sought after, naturally produced industrial oils in the marketplace (castor oil). Furthermore, proteomic strategies have already been developed for analysis of proteins derived from the ER of castor bean (Maltman et al, 2002), and these studies could be greatly aided by studying specific protein complexes that might be associated with key proteins known to be involved in castor oil biosynthesis (e.g. oleoyl-PC D 12 -hydroxylase, DGAT2).…”