Nuclear localization of Munc18-1 (p67) in the adult rat brain and PC12 cells

Abstract

Munc18-1, also referred to as p67, co-purifies with Cdk5 and has an important role in neurotransmitter release. The role of Munc18-1 for functional connectivity of the nervous system was demonstrated by gene knockout experiments in mice, wherein accumulation of neurotransmitter and silencing of synaptic activity was observed. Our earlier studies have shown that both Munc18-1 and Cdk5 co-purify and co-localize with cytoskeletal components, implying that apart from having a regulatory role in vesicle docking and fusion, Munc18-1 could also affect the dynamics of neuronal cytoskeleton. In the present study we have shown the presence of Munc18-1 in nuclear rich fraction from rat brain and confirmed the nuclear localization of this protein in PC12 cells and adult rat brain neurons by immunofluorescence and immunoelectron microscopy. We also demonstrate the binding of Munc18-1 to double stranded (ds) DNA. The ability of Munc18-1 to bind dsDNA, albeit the lack of DNA binding domains, suggests that the binding may be mediated through protein–protein interaction through some other DNA-binding proteins. The presence of both nuclear import and export signals in Munc18-1 primary structure corroborates its nuclear localization and makes it a putative shuttle protein between nuclear and cytoplasmic compartments, the precise physiological relevance of which needs to be elucidated.

Introduction

Munc18-1 (nSec1, Rab-Sec1) (for review see Toonen, 2003) is a neuron-specific protein, known to play a role in neurotransmitter release by mediating docking and fusion of synaptic vesicles to pre-synaptic membranes. Munc18-1 also referred to as p67 has been reported to co-purify with Cdc2-like kinase (later renamed as Cdk5) from the nervous system (Shetty et al., 1993, Shetty et al., 1995). An interaction of Cdk5 with Munc18-1 and resulting phosphorylation of Munc18-1 by Cdk5 have been demonstrated (de Vries et al., 2000). Munc18-1 has also been implicated in membrane fusion events required for neurite elaboration (Steiner et al., 2002). Further, in situ hybridization and immunocytochemical analysis of trigeminal ganglia and hippocampal neurons showed neuronal specificity of Munc18-1 and its rich distribution to axons (Shetty et al., 1995). In a recent study, we have shown co-localization and purification of Munc18-1 and Cdk5 with cytoskeletal components (Bhaskar et al., 2004). These observations suggest a role for Munc18-1, in association with Cdk5, in the structure and function of axons (Bhaskar et al., 2004, Shetty et al., 1995).

Munc18-1 plays a key role in vesicle secretion; however, there have been conflicting reports on the mechanism. Whereas sec1p, a t-SNARE binding protein, and its homologues are necessary for membrane trafficking and final stages of protein secretion, high affinity binding between Munc18-1 and syntaxin inhibits association of vesicle SNARES with syntaxin (Gengyo-Ando et al., 1993, Graham and Emr, 1991, Hata et al., 1993, Hosono et al., 1992, Hutchison, 2002), suggesting an inhibitory role for Munc18-1 in vesicle secretion. Conversely, Verhage et al. (2000) showed that deletion of Munc18-1 led to complete loss of neurotransmitter secretion from synaptic vesicle during development, suggesting a facilitatory role for Munc18-1. However, this did not prevent normal brain assembly, including formation of layered structures, fiber pathways, and morphologically defined synapses (Verhage et al., 2000). This study identified Munc18-1 as an upstream essential protein in neurotransmitter release, the ablation of which rendered the brain synaptically silent and caused the death of mutants in utero. Munc18-2 and Munc18-3, two isoforms of Munc18, have been reported in non-neuronal tissues and are presumed to be involved in cell secretion, although other functions have not been excluded (Tellam et al., 1995). Munc18 proteins respond to cell signaling, as they are targets of protein kinases and phosphatases (de Vries et al., 2000, Fletcher et al., 1999, Fujita et al., 1996).

Other than two major cellular functions of Munc18-1, cDNA derived amino acid sequence illustrated certain unique characteristics suggestive of potential additional cellular function(s). There are two clusters of basic amino acids at residue # 20–31 and # 457–467 that are characteristic of bipartite nuclear localization signal (NLS) sequence, implying the transport of Munc18-1 into the nucleus (Table 1). On the other hand, presence of a cluster of hydrophobic amino acids at residue # 405–414 in Munc18-1 (Table 1), representing the nuclear export signal (NES), is suggestive of its ability to be exported out of the nucleus. Earlier reports demonstrating that Munc18-1 exists as a component of multimeric complex with Cdk5 (Veeranna et al., 1997) and that Cdks translocate to nuclear compartment made it an interesting proposition to look for additional roles for this protein. The present study was designed to establish the physical evidence for the nuclear localization of Munc18-1 in neural tissue. We demonstrate here that Munc18-1 localizes to the nucleus of neurons of adult rat brain and PC12 cells. Besides, Munc18-1 was present in neuronal but not glial nuclear preparations. Munc18-1 was shown to bind double stranded DNA with strong affinity on a DNA-affinity column.