Proteomic dissection of the Arabidopsis Golgi and trans-Golgi network

Abstract

The plant Golgi apparatus and trans-Golgi network are major endomembrane trafficking hubs within the plant cell and are involved in a diverse and vital series of functions to maintain plant growth and development. Recently, a series of disparate technical approaches have been used to isolate and characterize components of these complex organelles by mass spectrometry in the model plant Arabidopsis thaliana. Collectively, these studies have increased the number of Golgi and vesicular localized proteins identified by mass spectrometry to nearly 500 proteins. We have sought to provide a brief overview of these technical approaches and bring the datasets together to examine how they can reveal insights into the secretory pathway.

Keywords: Arabidopsis; Golgi; LOPIT; SYP61; free-flow electrophoresis; proteomics; trans-Golgi network.

FIGURE 1

Overview of the three different techniques employed in proteomic characterization of the Arabidopsis Golgi and TGN. (A) Clustered proteins in LOPIT studies were assigned to the Golgi according to co-clustering with known and predicted Golgi marker proteins (for details, see Dunkley et al., 2004, 2006; Nikolovski et al., 2012). (B) FFE purified fractions were estimated at ca. 80% purity according to the proportion of previously localized Golgi proteins and contaminants present in each fraction; based on experimental data in SUBA (Heazlewood et al., 2007; for details, see Parsons et al., 2012a,c). (C) Isolation of SYP61 vesicles by affinity purification. Successful removal of contaminants during immunoisolation was assayed by the presence of the ER/cis-Golgi marker, BiP, and the prevacuolar compartment marker SYP21 (for details, see Drakakaki et al., 2012; Parsons et al., 2012c).