Subcellular Localization of Class II HDAs Figure 7. Nuclear localization and export signals of HDA15. A. Schematic representation of HDA15 is shown with its conserved histone deacetylase domain (HD), RanBP-type zinc finger (ZnF), nuclear localization signals (NLS), and nuclear export signal (NES). HDA15 contains three NLS signals, one classical par4 type NLS near the N-terminal and an overlapping bipartite NLS near the C-terminal end hereby jointly deleted as NLS2, and an NES stationed before the bipartite NLS near the carboxyl end. Numbers 1-6 illustrate the different truncated constructs of HDA15 where varying combinations of NLS and NES signals were deleted, shown here as dash lines. B. To determine if HDA15 depends on its NLS and NES for its subcellular localization, these predicted signals were truncated out of the HDA15-YFP and transiently expressed in protoplasts. Both NLS1 and NLS2 functionally direct the nuclear localization of HDA15 and remains active even in the absence of the other. However, the deletion of both NLS renders HDA15 cytoplasmic indicating the functionality of its NES. This suggests that HDA15 can translocate in and out of the nucleus and potentially undergo nucleocytoplasmic shuttling. doi:10.1371/journal.pone.0030846.g007 phosphorylate Class IIa HDACs. These include Ca2+/calmodulin- dependent kinases [61,64], salt-inducible kinases [65,66], protein kinase D [67–69], microtubule affinity-regulating kinases [70,71], and checkpoint kinase-1 [72]. It is highly likely that microtubule affinity-regulating kinases may be involved in phosphorylating 14-3- 3 binding sites with HDA5 considering that it localizes in the cytoskeletal region. Nevertheless, more studies are needed to elucidate further the mechanisms governing HDA5 and 14-3-3 interaction as well as the exact substrates and function of HDA5 in plants. HDA8 prominently occupies the cytoplasm as well as peripheral areas of the nucleus. However, subcellular markers including ER, golgi, trans-golgi network, prevacuolar compartment, and mito- chondria staining failed to overlap with its pertinent localization suggesting HDA8 to be suspended in the cytoplasma. Compared to all the Class II HDAs in Arabidopsis, it is the sole histone deacetylase devoid of an NLS but with a NES imprinted along the histone deacetylase region, which is similar to the conserved HDA domain of human Class IIa HDACs. In the absence of an NLS, HDA8 may need to associate with other proteins that would chaperone its translocation to undergo nuclear import. Although its conserved histone deacetylase domain is homologous to Class II HDAs compared to the other members of the RPD3/HDA1-like superfamily, it is more probable that its function is stationed in the PLoS ONE | www.plosone.org 9 February 2012 | Volume 7 | Issue 2 | e30846