Quantitative analysis of subcellular distribution of the SUMO conjugation system by confocal microscopy imaging

Different studies point to an enrichment in SUMO conjugation in the cell nucleus, although non-nuclear SUMO targets also exist. In general, the study of subcellular localization of proteins is essential for understanding their function within a cell. Fluorescence microscopy is a powerful tool for studying subcellular protein partitioning in living cells, since fluorescent proteins can be fused to proteins of interest to determine their localization. Subcellular distribution of proteins can be influenced by binding to other biomolecules and by posttranslational modifications. Sometimes these changes affect only a portion of the protein pool or have a partial effect, and a quantitative evaluation of fluorescence images is required to identify protein redistribution among subcellular compartments. In order to obtain accurate data about the relative subcellular distribution of SUMO conjugation machinery members, and to identify the molecular determinants involved in their localization, we have applied quantitative confocal microscopy imaging. In this chapter, we will describe the fluorescent protein fusions used in these experiments, and how to measure, evaluate, and compare average fluorescence intensities in cellular compartments by image-based analysis. We show the distribution of some components of the Arabidopsis SUMOylation machinery in epidermal onion cells and how they change their distribution in the presence of interacting partners or even when its activity is affected.This work was supported by the European Research Council (grant ERC-2007-StG-205927) and Departament d’Innovació, Universitats i Empresa from the Generalitat de Catalunya (Xarxa de Referència en Biotecnologia and 2014SGR447). A.M. was supported by predoctoral fellowships FPU12/05292. This article is based upon work from COST Action (PROTEOSTASIS BM1307), supported by COST (European Cooperation in Science and Technology).Peer reviewe

Highly functionalized terpyridines as competitive inhibitors of AKAP-PKA interactions.

A good fit: Interactions between A-kinase anchoring proteins (AKAPs) and protein kinaseA (PKA) play key roles in a plethora of physiologically relevant processes whose dysregulation causes or is associated with diseases such as heart failure. Terpyridines have been developed as α-helix mimetics for the inhibition of such interactions and are the first biologically active, nonpeptidic compounds that block the AKAP binding site of PKA. © 2013 The Authors. Published by Wiley-VCH Verlag GmbH and Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited

Importin α/β mediates nuclear import of individual SUMO E1 subunits and of the holo-enzyme

SUMOylation is an essential modification that regulates predominantly nuclear proteins. Here we describe two pathways for the generation of nuclear SUMO E1 enzyme, import of individual subunits, and transport of the holo-enzyme. The NLS in Uba2 is required for transport of the complex; the c-Myc–like NLS in Aos1 functions only in the free subunit