Proteomic analysis of the Endoplasmic Reticulum of developing castor bean seeds - Testing the feasibility of ER membrane topology elucidation using a sample of purified ER

Abstract

An Endoplasmic Reticulum (ER) membrane topology elucidation experiment involving ER purified according to Simon et al. [2] was thought to identify yet unknown, tri‐ricinoleate biosynthetic enzymes via elucidation of membrane topology and identification of membrane associated proteins. This thesis aims at testing the feasibility of an ER membrane topology elucidation experiment using purified castor bean seed ER prepared according to Simon et al [2].
In a bioinformatics approach, proteins identified in a Multidimensional Protein Identification Technology (MudPIT) experiment of an ER preparation from castor bean seeds were compared to predicted ER localised proteins, thus validating the prediction algorithms, as well as verifying possible contaminations of the ER preparation. Antibodies against identified organelle marker proteins were used in quantitative Western Blot analysis to confirm the degree of purification and contamination. Relative comparison of developing endosperm castor bean ER (dER) preparation with a developing endosperm homogenate showed that, when using anti‐oleate Δ 12‐hydroxylase
(an ER marker protein) antibodies, purification of 58x ER had occurred. When using an anti‐VDAC (a mitochondrial marker protein) antibody, a relative quantitation showed that VDAC was 10 x more concentrated in a dER prep than in homogenate. Unlike in previous experiments where mitochondria were purified and 327 mitochondrial proteins were discovered, only 13 could be identified in a MudPIT experiment on Ricinus communis ER, thus it can be concluded that mitochondria did not co‐purify with dER. The ER preparation was then imaged under a Scanning Electron Microscope. 3 different visualisation techniques were developed to ensure that pictures reflect the original ER preparation state. Vesicles were far more numerous than complex structures. Taking into account the thermodynamics of membranes, it can be said the ER preparation by Simon
et al. [2] does not consist of whole ER, but rather ER vesicles.