Proteomic Characterization of Golgi Membranes Enriched from Arabidopsis Suspension Cell Cultures
The plant Golgi apparatus has a central role in the secretory pathway and is the principal site within the cell for the assembly and processing of macromolecules. The stacked membrane structure of the Golgi apparatus along with its interactions with the cytoskeleton and endoplasmic reticulum has historically made the isolation and purification of this organelle difficult. Density centrifugation has typically been used to enrich Golgi membranes from plant microsomal preparations, and aside from minor adaptations, the approach is still widely employed. Here we outline the enrichment of Golgi membranes from an Arabidopsis cell suspension culture that can be used to investigate the proteome of this organelle. We also provide a useful workflow for the examination of proteomic data as the result of multiple analyses. Finally, we highlight a simple technique to validate the subcellular localization of proteins by fluorescent tags after their identification by tandem mass spectrometry.
Key wordsGolgi apparatus Density gradient centrifugation Subcellular localization Fluorescent protein
This work was funded by grants from the Australia Research Council (ARC) to the ARC Centre of Excellence in Plant Cell Walls [CE110001007] and the US Department of Energy, Office of Science, Office of Biological and Environmental Research, through contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the US Department of Energy. J.L.H. is supported by an ARC Future Fellowship [FT130101165]. S.F.H. was supported by a research grant [VKR023371] from VILLUM FONDEN. We also wish to thank the UC Davis Proteomics Core Facility for sample analysis.
- 9.Morré DJ, Mollenhauer HH (1974) The endomembrane concept: a functional integration of endoplasmic reticulum and Golgi apparatus. In: Robards AW (ed) Dynamic aspects of plant infrastructure. McGraw-Hill, New York, pp 84–137Google Scholar
- 17.Parsons HT, González Fernández-Niño SM, Heazlewood JL (2014) Separation of the plant Golgi apparatus and endoplasmic reticulum by free-flow electrophoresis. In: Jorrín Novo JV, Komatsu S, Weckwerth W, Weinkoop S (eds) Plant proteomics: methods and protocols, vol 1072, 2nd edn. Humana Press, New York, pp 527–539CrossRefGoogle Scholar