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Proteomic dissection of the Arabidopsis Golgi and trans-Golgi network.

Parsons HT, Drakakaki G, Heazlewood JL - Front Plant Sci (2013)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant and Environmental Sciences, University of Copenhagen Copenhagen, Denmark.

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.

No MeSH data available.


Related in: MedlinePlus

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).
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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).

Mentions: Initial attempts to characterize the Arabidopsis Golgi by mass spectrometry were undertaken nearly a decade ago with the aim of distinguishing between ER- and Golgi-resident proteins (Dunkley et al., 2004). The LOPIT approach involves quantitative mass spectrometry of proteins labeled with isotope tags. A cell homogenate separated along a linear gradient is fractionated and pairwise comparisons of fractions allow abundance ratios of isotope masses to be calculated for each protein. Proteins physically located in the same compartment will have similar ratios and so cluster together during partial least squares discriminant analysis (Figure 1). Using LOPIT, 89 proteins were initially localized to the Golgi (Dunkley et al., 2006) but the requirement that proteins carry all four tags limited the number of proteins for which a statistically credible localization could be assigned. Recent reanalysis and analysis of existing and new datasets, incorporating values for “missing” tags assigned using partial least squares regression models and training sets based on fully tagged proteins, enabled the collective localization of 204 proteins to the Golgi/TGN (Dunkley et al., 2006; Nikolovski et al., 2012).


Proteomic dissection of the Arabidopsis Golgi and trans-Golgi network.

Parsons HT, Drakakaki G, Heazlewood JL - Front Plant Sci (2013)

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).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3539648&req=5

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).
Mentions: Initial attempts to characterize the Arabidopsis Golgi by mass spectrometry were undertaken nearly a decade ago with the aim of distinguishing between ER- and Golgi-resident proteins (Dunkley et al., 2004). The LOPIT approach involves quantitative mass spectrometry of proteins labeled with isotope tags. A cell homogenate separated along a linear gradient is fractionated and pairwise comparisons of fractions allow abundance ratios of isotope masses to be calculated for each protein. Proteins physically located in the same compartment will have similar ratios and so cluster together during partial least squares discriminant analysis (Figure 1). Using LOPIT, 89 proteins were initially localized to the Golgi (Dunkley et al., 2006) but the requirement that proteins carry all four tags limited the number of proteins for which a statistically credible localization could be assigned. Recent reanalysis and analysis of existing and new datasets, incorporating values for “missing” tags assigned using partial least squares regression models and training sets based on fully tagged proteins, enabled the collective localization of 204 proteins to the Golgi/TGN (Dunkley et al., 2006; Nikolovski et al., 2012).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant and Environmental Sciences, University of Copenhagen Copenhagen, Denmark.

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.

No MeSH data available.


Related in: MedlinePlus