Septin 3 (G‐septin) is a developmentally regulated phosphoprotein enriched in presynaptic nerve terminals

Abstract

The septins are GTPase enzymes with multiple roles in cytokinesis, cell polarity or exocytosis. The proteins from the mammalian septin genes are called Sept1-10. Most are expressed in multiple tissues, but the mRNA for Sept5 (CDCrel-1) and Sept3 (G-septin) appear to be primarily expressed in brain. Sept3 is phosphorylated by cGMP-dependent protein kinase I (PKG-I) and the cGMP/PKG pathway is involved in presynaptic plasticity. Therefore to determine whether Sept3 specifically associates with neurones and nerve terminals we investigated its distribution in rat brain and neuronal cultures. Sept3 protein was detected only in brain by immunoblot, but not in 12 other tissues examined. Levels were high in all adult brain regions, and reduced in those enriched in white matter. Expression was developmentally regulated, being absent in the early embryo, low in late embryonic rat brain and increasing after birth. Like dynamin I, Sept3 was specifically enriched in synaptosomes compared with whole brain, and was only found in a peripheral membrane extract and not in the soluble or membrane extracts. Sept3 was particularly abundant in mossy fibre nerve terminals in the hippocampus. In primary cultured hippocampal neurones Sept3 immunoreactivity was punctate in neurites and predominantly localized to presynaptic terminals, strongly colocalizing with synaptophysin and dynamin I. The specific nerve terminal localization was confirmed by immunogold electron microscopy. Together this shows that Sept3 is a neurone-specific protein highly enriched in nerve terminals which supports a secretory role in synaptic vesicle recycling. Keywords: cGMP, G-septin, PKG, protein phosphorylation, septin 3, synaptosomes. The septins are a multigene family in mammals comprising 10 genes, producing a variety of protein products by further alternative splicing. The proteins are named Sept1-10 and are GTPase enzymes of around 40-70 kDa (Macara et al. 2002;Surka et al. 2002). The GTPase domain comprises the bulk of the sequence and no other large domains are found, although a few short functional motifs are known (Saraste et al. 1990;Zhang et al. 1999). The septins have multiple cellular functions, primarily playing an essential role in cytokinesis (Kinoshita et al. 1997;Hsu et al. 1998;Xie et al. 1999;Surka et al. 2002). In non-mitotic cells septins appear to assemble into filaments and/or associate with the plasma membrane Fung and Scheller 1999;Zhang et al. 1999). Groups of different septins also assemble as filaments in vitro (Field et al. 1996;Hsu et al. 1998;Kinoshita 2003). The filaments appear to be strongly associated with either actin (Kinoshita et al. 1997;Kinoshita et al. 2002) or tubulin networks (Surka et al. 2002), or in some cases with both (Surka et al. 2002). The filaments can self-assemble into higher-order structures to form

The Role of cGMP-Dependent Signaling Pathway in Synaptic Vesicle Cycle at the Frog Motor Nerve Terminals

“…Another possibility relates to the phosphorylation of G-septin by PKG. Septins are a family of GTPase enzymes, primarily concentrated in the nerve terminal (Xue et al, 2004), which are able to assemble into polymers and to act as diffusion barriers between different subcellular compartments. Septins might therefore tether SVs, including the vesicles of mobilization pool, to the region near the active zones and thus prevent their trafficking to the reserve pool.…”

“…Most of the observed effects of cGMP are mediated by protein kinase G (PKG) activation (Wang et al, 2005). Some proteins participating in the processes of exo/endocytosis and SVs trafficking can be phosphorylated by PKG (Xue et al, 2004). In hippocampal neurons PKG mediates the presynaptic component of long-term depression and long-term potentiation (Kleppisch et al, 2003, Stanton et al, 2003.…”

Cyclin-Dependent Kinase 5 Phosphorylation of Human Septin SEPT5 (hCDCrel-1) Modulates Exocytosis

“…Among several putative proteins we extracted was human septin 5 (SEPT5), previously called hCDCrel-1 (Macara et al, 2002), one of many septins spe-cific to the brain, which colocalizes with synaptic vesicles and is involved in exocytosis (Xue et al, 2004;Beites et al, 2005). Previous studies relating septin 5 to synaptic vesicle proteins and exocytosis have been performed with rat septin 5 (Beites et al, 1999).…”

Septin 11 Is Present in GABAergic Synapses and Plays a Functional Role in the Cytoarchitecture of Neurons and GABAergic Synaptic Connectivity

“…Septins 3, 5, 6, and 7 are localized in the presynaptic terminals, frequently associated with synaptic vesicles (6,16,17). In neurons, septin 11 forms heterooligomeric complexes with septin 7 and septin 5 (9, 18).…”