In vivo membrane trafficking role for an insect N-ethylmaleimide-sensitive factor which is developmentally regulated in endocrine cells

摘要

The hexameric ATPase, N-ethylmaleimide-sensitive factor (NSF) is implicated in the release of neurotransmitters and in mediating fusion between intracellular membranes. Due to the conservation of proteins in constitutive and regulated membrane fusion reactions, NSF and its downstream targets have been predicted also to participate in fusion reactions underlying endocrine function, but there is little experimental evidence to support such a role for NSF in insect neuroendocrine secretion. Here we have characterized the NSF orthologue (MsNSF) from the endocrine model for development Manduca sexta. MsNSF is developmentally regulated in endocrine organs of the protocerebral complex. Enrichment of MsNSF in corpora cardiaca (CC) and not in corpora allata (CA) indicates that it might play a preferential role in releasing hormones produced in CC. Endocrine/paracrine cells of the enteric system in ill. sexta exhibit selective MsNSF enrichment. Together the data point to a more selective participation of MsNSF in development of ill. sexta by its involvement in a subset of factors, whereas other as-yet-unidentified homolog(s) might regulate secretion from CA and a large set of endocrine/paracrine cells. We further characterized the in vivo role of MsNSF by heterologous expression. In contrast to vertebrate NSF, MsNSF is functional in yeast membrane fusion in vivo. MsNSF rectifies defects in SEC18 (yeast NSF homologue) at nearly all discernible steps where Sec18p has been implicated in the biosynthetic route. This underscores the utility of our approach to delineate functional roles for proteins from systems that are not currently amenable to in vitro reconstitution.