Nucleoplasmic/nucleolar translocation and identification of a nuclear localization signal (NLS) in Dictyostelium BAF60a/SMARCD1 homologue Snf12

Abstract

Dictyostelium is a model eukaryote for the study of several cellular processes; however, comparatively little is known about its nucleolus. Identification of nucleolar proteins is key to understanding this nuclear subcompartment, but only four have been identified in Dictyostelium. As discussed in this article, a potential relationship between nucleolar NumA1 and BAF60a/SMARCD1 suggested BAF60a may also reside in the nucleolus. Here, we identify BAF60a homologue Snf12 as the fifth nucleolar protein in Dictyostelium. Immunolocalization experiments demonstrate that Snf12 is nucleoplasmic, but translocates to nucleoli upon actinomycin-D-induced transcription inhibition (0.05 mg/mL, 4 h). Translocation was accompanied by a microtubule-independent protrusion of nucleolar Snf12 regions from the nucleus followed by detection of Snf12 in cytoplasmic circles for at least 48 h. Residues (372)KRKR(375) are both necessary and sufficient for nucleoplasmic localization of Snf12 and represent a functional nuclear localization signal (NLS), similar to recently identified NLSs in other Dictyostelium proteins. Since nucleolar and nucleoplasmic proteins redistribute during mitosis, we investigated Snf12 dynamics during this time. Dictyostelium undergoes closed mitosis, meaning its nuclear envelope remains intact. Despite this, during metaphase and anaphase Snf12 redistributed throughout the cytoplasm before reaccumulating in the nucleus during telophase, unlike the previously reported nucleoplasmic redistribution of nucleolar NumA1. The nuclear exit of Snf12 was independent of its putative nuclear export signal and not inhibited by exportin inhibition, suggesting that the redistribution of nuclear proteins during mitosis in Dictyostelium is mediated by other mechanisms. Snf12 is the second Dictyostelium nucleolar protein for which its dynamics during mitosis have been investigated.

Evidence for nucleolar subcompartments in Dictyostelium

“…Snf12 localizes to a portion of the nucleolus GFP-Snf12 was recently shown to localize predominately to the nucleoplasm and, in 20% of cells, also to the nucleolus [17]. In order to determine if Snf12 localizes throughout the entire nucleolus C-GFP-Snf12 was colocalized with the nucleolar marker NumA1.…”

“…Anti-NumA1, anti-CBP4a, and anti-Snf12 were produced and verified as previously described [15][16][17]19]. Cell lines expressing either C-GFP-Snf12 or GFP-NLS-3 (GFP-KRKR) from Snf12 were made and verified as previously described [17].…”

“…Cell lines expressing either C-GFP-Snf12 or GFP-NLS-3 (GFP-KRKR) from Snf12 were made and verified as previously described [17]. Cell lines expressing either C-GFP-eIF6 or C-GFP-Bud31 were made by amplifying full length eIF6 or Bud31 via PCR using total cDNA as template as previously described [16].…”

“…Calcium-binding protein 4a (CBP4a) and forkhead-associated kinase A (fhkA) are two recently identified proteins in Dictyostelium that localize to different nucleolar locales; FhkA localizes around the nucleolar periphery while CBP4a resides within the nucleoplasm [15]. BAF60a/SMARCD1 homologue Snf12 and NumA1, two nucleolar proteins linked to cell cycle in Dictyostelium, localize differently since Snf12 localizes to a smaller area of the nucleolus than NumA1 [16,17]. If, as this work suggests, functional subcompartments do exist, these and other nucleolar proteins should localize to different regions of the nucleolus, thereby delineating some of the nucleolar subcompartment locales.…”

A new HECT ubiquitin ligase regulating chemotaxis and development in Dictyostelium discoideum

“…The fusion protein is 70 kDa in size, thus nuclear enrichment cannot be due to passive diffusion. Since the plasmid constructs do not contain nuclear localization signals typical of Dictyostelium (Catalano and O'Day, 2012), it is possible that the HECT domain is co-transported to the nucleus bound to a potential substrate. To what extent the nuclear localization is an artefact of the isolated HECT fragment is an open question.…”