Human promyelocytic leukemia protein is targeted to distinct subnuclear domains in plant nuclei and colocalizes with nucleolar constituents in a SUMO‐dependent manner

Abstract

Eukaryotic nuclei are subdivided into subnuclear structures. Among the most prominent of these structures are the nucleolus and the PML nuclear bodies (PML‐NBs). PML‐NBs are spherical multiprotein aggregates of varying size localized in the interchromosomal area. PML‐NB formation is dependent on the presence of the promyelocytic leukemia protein (PML) as well as on post‐translational modification of core components by covalent attachment of the small ubiquitin‐like modifier (SUMO). So far, PML‐NBs as well as PML have been described in mammalian cells only, whereas no orthologs are known in the plant kingdom. In order to investigate conserved mechanisms in PML targeting, we expressed human PML (hPML) fused to the GFP in Nicotiana benthamiana. Using confocal laser scanning microscopy and coimmunoprecipitation followed by mass spectrometric analysis, we found the fusion protein in association with nucleolar constituents. Importantly, mutants of hPML, which are no longer SUMOylated, showed altered localizations, implying SUMO‐dependent targeting of hPML in plants as has previously been shown for mammalian cells. Interestingly, in the presence of proteasome inhibitors, hPML could also be found in the nucleolus of mammalian cells suggesting conserved targeting mechanisms of PML across kingdoms. Finally, Solanum tuberosum COP1, a proposed PML‐like protein from plants, was fused to the red fluorescent protein (RFP) and coexpressed with hPML::eGFP. Microscopic analysis confirmed the localization of COP1::RFP in nuclear speckles. However, hPML::eGFP did not colocalize with COP1::RFP. Hence, we conclude that plants do not possess specialized PML‐NBs, but that their functions may be covered by other subnuclear structures like the nucleolus. Database Proteomics data have been deposited to the ProteomeXchange Consortium with the identifier PXD004254.

ChMob2 binds to ChCbk1 and promotes virulence and conidiation of the fungal pathogen Colletotrichum higginsianum

“…Purified proteins were eluted from the beads using two times 50 μl of 0.2 M glycine-HCl buffer (pH 2.5) and then neutralized with 10 μl of 1 M Tris–HCl (pH 10.4). Tryptic digestion of the eluted samples, their processing and Nano-LC-MS/MS analysis was performed as described [64]. Raw data files were evaluated using Peaks7 (Bioinformatics Solutions Inc., Waterloo, ON, Canada) and a Colletotrichum higginsianum proteome database [26].…”