RAE-1, a Novel PHR Binding Protein, Is Required for Axon Termination and Synapse Formation inCaenorhabditis elegans

Abstract

Previous studies in C. elegans showed that RPM-1 (Regulator of Presynaptic Morphology-1) regulates axon termination and synapse formation. In order to understand the mechanism of how rpm-1 functions, we have used mass spectrometry to identify RPM-1 binding proteins, and have identified RAE-1 (RNA Export protein-1) as an evolutionarily conserved binding partner. We define a RAE-1 binding region in RPM-1, and show that this binding interaction is conserved and also occurs between Rae1 and the human ortholog of RPM-1 called Pam. rae-1 loss of function causes similar axon and synapse defects, and synergizes genetically with two other RPM-1 binding proteins, GLO-4 and FSN-1. Further, we show that RAE-1 colocalizes with RPM-1 in neurons, and that rae-1 functions downstream of rpm-1. These studies establish a novel postmitotic function for rae-1 in neuronal development.

Identification of a Peptide Inhibitor of the RPM-1·FSN-1 Ubiquitin Ligase Complex

“…Thus, although RAE-1 and FSN-1 are likely to be in close physical proximity, FSN-1 is unlikely to act as an adaptor for recruitment of RAE-1 into RPM-1 protein complexes and vice versa. Our biochemical results are consistent with prior genetic and proteomic results, which showed that RAE-1 is not a target of RPM-1 ubiquitin ligase activity (28,33).…”

“…Note that FLAG-FSN-1 coprecipitated exclusively with domain 5 of RPM-1 (GFP-D5) (top panel). Consistent with a previous study, FLAG-RAE-1 also coprecipitated with GFP-RPM-1 domain 5 (GFP-D5) (top panel, last lane) (28). Shown are representatives of experiments that were independently performed at least three times.…”

“…We found that five highly conserved motifs within FBD1 are required for binding to FSN-1. FBD1 is contained within a larger domain we previously showed binds to RAE-1 (28). Our results here show that FSN-1 relies upon a different binding site in RPM-1 than RAE-1 (Figs.…”

“…We previously showed that D5 is sufficient for binding to RAE-1, so we also refer to this domain as the RAE-1 binding domain (Fig. 1, A and B) (28). We noted that coexpression of D5 with FSN-1 consistently resulted in increased expression of FSN-1 (Figs.…”

“…If RIP inhibits the RPM-1⅐FSN-1 complex, we expected transgenic overexpression of RIP to yield phenotypes that were similar to fsn-1 (lf) mutations. Notably, we did not expect RIP overexpression to yield phenotypes that occurred with the same expressivity as rpm-1 (lf) because RPM-1 functions through several FSN-1 independent mechanisms including the GLO Rab pathway, the microtubule-binding protein RAE-1, the phosphatase PPM-2, and the ANC-1/␤-catenin pathway (12,28,31,32). We generated transgenic animals that overexpressed FLAG epitopetagged RIP by injecting PCR product at relatively high concentrations (10 ng/l).…”