Functional regions of the presynaptic cytomatrix protein bassoon: significance for synaptic targeting and cytomatrix anchoring

Abstract

Exocytosis of neurotransmitter from synaptic vesicles is restricted to specialized sites of the presynaptic plasma membrane called active zones. A complex cytomatrix of proteins exclusively assembled at active zones, the CAZ, is thought to form a molecular scaffold that organizes neurotransmitter release sites. Here, we have analyzed synaptic targeting and cytomatrix association of Bassoon, a major scaffolding protein of the CAZ. By combining immunocytochemistry and transfection of cultured hippocampal neurons, we show that the central portion of Bassoon is crucially involved in synaptic targeting and CAZ association. An N-terminal region harbors a distinct capacity for N-myristoylation-dependent targeting to synaptic vesicle clusters, but is not incorporated into the CAZ. Our data provide the first experimental evidence for the existence of distinct functional regions in Bassoon and suggest that a centrally located CAZ targeting function may be complemented by an N-terminal capacity for targeting to membrane-bounded synaptic organelles.

Introduction

Active zones are specialized regions of the presynaptic plasma membrane where synaptic vesicles (SVs) dock and fuse to release neurotransmitter. A key signature of active zones is a dense meshwork of cytoskeletal filaments originating at the presynaptic plasma membrane and extending into the SV domain Burns and Augustine 1995, Hirokawa et al 1989, Landis et al 1988. This meshwork, termed cytomatrix at the active zone (CAZ) or presynaptic particle web, may serve pivotal synaptic functions, including anchoring and mobilization of SVs and spatial restriction of SV exocytosis to and assembling presynaptic molecules at the active zone Dresbach et al 2001, Garner et al 2000, Phillips et al 2001, Zamorano and Garner 2001.

Five CAZ-specific proteins have been identified to date, including Munc13 (Betz et al., 2001), RIM (Wang et al., 1997), CAST (Ohtsuka et al., 2002), Bassoon, and Piccolo/Aczonin. The latter two proteins are giant CAZ components of 420 and 530 kDa, respectively, present at both excitatory and inhibitory synapses Cases-Langhoff et al 1996, Fenster et al 2000, Richter et al 1999, tom Dieck et al 1998, Wang et al 1999. They are members of a new family of multidomain proteins sharing regions of high sequence similarity, including two double zinc finger domains in an N-terminal region and three predicted coiled-coil domains in a central region of each protein Fenster et al 2000, tom Dieck et al 1998, Wang et al 1999, Winter et al 1999. Both proteins are tightly anchored to the subsynaptic cytoskeleton as judged by their resistance to detergent extraction from synaptosomal fractions Cases-Langhoff et al 1996, tom Dieck et al 1998. Long regions of Bassoon and Piccolo share features of extended cytoskeletal proteins and it is possible that they span the entire CAZ extending from the presynaptic plasma membrane deeply into the presynaptic space Brodin et al 1997, Dresbach et al 2001. Their multidomain structure and enormous size suggest that these proteins can act as major scaffolding proteins of the CAZ. Moreover, they are among the earliest proteins to be incorporated into nascent synapses, consistent with a role in active zone assembly Vardinon-Friedman et al 2000, Zhai et al 2000, Zhai et al 2001.

Despite their exquisite localization in the CAZ and putative role in active zone assembly, little is known about the mechanisms of CAZ assembly and of targeting of cytomatrix proteins to active zones. In particular, because Bassoon and Piccolo have little homology to any other known protein it is difficult to extract information on putative targeting signals from their primary structure. We have recently found Bassoon and Piccolo associated with a novel class of organelles, which may be involved in transport of CAZ proteins (Zhai et al., 2001). However, questions concerning the regions of these proteins that may mediate presynaptic targeting and the mechanisms underlying the particular specificity of CAZ targeting have remained open. Here we address these questions by analyzing subcellular targeting of recombinant Bassoon in cultured hippocampal neurons. Our data show that the central region of Bassoon is essential for its association with the CAZ. Moreover our data provide evidence that an N-terminal region of Bassoon can be targeted independently to presynaptic boutons. This latter process critically depends on the presence of a functional myristoylation site at the amino terminus.