Subcellular Localization of Class II HDAs Figure 5. Organelle markers and Class II HDA localization. Specific organelle markers fused with RFP and mitoTracker stain were used to identify the subcellular localization of Class II HDAs. The ER marker, HDEL, overlaps with the localization of HDA5-GFP. Although HDA8 predominantly abounds in the cytoplasm, partial nuclear localization was observed at the surrounding areas of the nucleus using VirD2NLS as nuclear marker. Moreover, HDA15 concentrates in a small spot inside the nucleus, potentially nucleolus. On the other hand, HDA14-YFP was confirmed to localize in the chloroplasts and mitochondria using mitoTracker. Protoplasts derived from Arabidopsis PSB-D lines were used for PEG transfection in HDA5, HDA8, and HDA15 while protoplasts from Arabidopsis leaves were utilized for the HDA14-YFP localization. Scale bars were calibrated to 10 mm. doi:10.1371/journal.pone.0030846.g005 which inactivates its catalytic activity as a histone deacetylase via subcellular compartmentalization. However, they can also target cytosolic substrates especially when bound to specific organelles. Plant Class II HDAs may exhibit similar functional regulation as well as versatile roles since all the Class II HDAs were observed to be cytoplasmic. HDA5 prominently localizes in the cytoplasm, lining the nucleus, in distinct web like structure resembling the cytoskel- eton, which was confirmed by the use of HDEL, an endoplasmic reticulum marker. The ER is composed of an extensive network of cisternae held together by the cytoskeleton. Studies by Mi and Puglielli [62] have implicated the presence of HATs and HDACs in the ER and Golgi describing the reversible acetylation of BACE1, a protease responsible for amyloid precursor protein cleavage, and LDL receptor, respectively. Although the HAT responsible for this occurrence has been determined, the identity of the HDAC still remains to be discovered. It is probable that the human orthologue of HDA5 could catalyze the deacetylation of these proteins. Furthermore, the dynamic movement of HDA5 along the cytoskeletal network in onion tissues implicates its potential function in tubulin deacetylation which may be similar to human HDAC6. Phylogenetic analysis conducted by Gregoretti et al. [63] proposes HDA5 to be the plant orthologue of mammalian HDAC6 which deacetylates alpha- tubulin similarly conserved in all eukaryotes. But unlike human HDAC6, which is exclusively cytoplasmic and depends on its own NLS and NES, HDA5 localizes both cytoplasm and nucleus and positively binds with 14-3-3 potentially for its nuclear export and cytoplasmic retention since it does not have any NES. Similar to the human Class IIa HDAC5, the inability of HDA5 to bind with 14-3-3 K and E indicates that specific kinases which are absent in yeast, are required to catalyze the phosphor- ylation of 14-3-3 for its binding to HDA5. In humans and other model organisms, five kinase groups have been shown to PLoS ONE | www.plosone.org 7 February 2012 | Volume 7 | Issue 2 | e30846