Isolation and proteomic analysis of the SYP61 compartment reveal its role in exocytic trafficking in Arabidopsis
Authors: Songqin Pan
Wilhelmina van de Ven
Brent Weatherly
Glenn Hicks
Junqi Wang
Natasha Raikhel
Liwen Jiang
Karin Schumacher
Nana F Keinath
Georgia Drakakaki
Yansong Miao
Journal: Cell Research (2012)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: endomembrane system; intracellular trafficking network; affinity purification; vesicles; SYP61
Affiliations: Department of Botany and Plant Sciences, Center for Plant Cell Biology, Institute for Integrative Genome Biology, 4119C Genomics Building, University of California Riverside, CA 92521, USA.

Abstract:  The endomembrane system is a complex and dynamic intracellular trafficking network. It is very challenging to track individual vesicles and their cargos in real time; however, affinity purification allows vesicles to be isolated in their natural state so that their constituent proteins can be identified. Pioneering this approach in plants, we isolated the SYP61 trans-Golgi network compartment and carried out a comprehensive proteomic analysis of its contents with only minimal interference from other organelles. The proteome of SYP61 revealed the association of proteins of unknown function that have previously not been ascribed to this compartment. We identified a complete SYP61 SNARE complex, including regulatory proteins and validated the proteome data by showing that several of these proteins associated with SYP61 in planta. We further identified the SYP121-complex and cellulose synthases, suggesting that SYP61 plays a role in the exocytic trafficking and the transport of cell wall components to the plasma membrane. The presence of proteins of unknown function in the SYP61 proteome including ECHIDNA offers the opportunity to identify novel trafficking components and cargos. The affinity purification of plant vesicles in their natural state provides a basis for further analysis and dissection of complex endomembrane networks. The approach is widely applicable and can afford the study of several vesicle populations in plants, which can be compared with the SYP61 vesicle proteome. 

Retracted: No

PubPeer comments:
 Western blot (boxed) in Fig. 1A looks strange. Even when contrast is stretched to maximum (analysis below), no background in the elution part shows up whatsoever. This control is important to demonstrate purity of TGN vesicles versus PVC vesicles, and all later conclusions from proteomic depend on sample purity. Could the authors please post the original blot, with a detectable background, in order to appreciate what has been loaded there?  , 
 Here the plot thickens. Again Figure 1A. Nine months ago I did not realize that the upper blot is also problematic (yellow boxes). It appears as the two bands are the same, just flipped horizontally. Image analysis (gradient map) highlights a remarkable symmetry of light dots (in yellow for gradient map) within the two bands. Minor differences are likely to be due to compression and or slightly different horizontal dimension. Red arrow indicate a sharp vertical edge, which would be the splice. Could the authors please comment and post the uncropped scan of the blot?  , 
 Again Figure 1A, thid time mid blot. Contrast enhancement highlights a putative splice (arrow). Could the authors please post the uncropped scan of the mid blot too?  , 
 Figure 1B panel (ii): Negative staining and immunolocalization of SYP61 in purified vesicles with a SYP61 antibody (image below). **No negative control present for this data.** The well known ability of nanogold particles to stick unspecifically on clumps of organic material that simply lays on (and protrudes from) the surface of a thin coated film did not concern the authors, who chose not to include a **mandatory** negative control.  ,