A series of in vivo
studies in a variety of organisms have found that the evolutionarily conserved PHR proteins, including Hiw (fly), Phr1 (mouse), RPM-1 (worm) and Esrom (zebrafish), regulate the development and repair of the nervous system1
. In Drosophila
mutants exhibit a marked synaptic terminal overgrowth at the larval neuromuscular junctions (NMJs) with an increased number of boutons and a decreased bouton size2,3
, and a similar anatomical overgrowth of the giant fiber–tergotrochanteral motoneuron synapse in the CNS4
. Aberrant synaptic morphology has also been identified in rpm
. In addition, disrupting the function of hiw
orthologs in worm, zebrafish and mouse results in prominent axon guidance defects5,7,9–11
. More recently, hiw
and their downstream target wallenda
(fly) or dlk
(worm) have been shown to regulate axonal regeneration after nerve injury12–14
. Thus, PHR proteins are important in a broad range of neuronal processes, including axon guidance, synaptic development and axonal regeneration. However, it is unclear how these diverse functions of PHR proteins are achieved. An important step toward answering this question is to understand the molecular mechanisms that regulate PHR proteins.
Hiw and its orthologs are enormous proteins that share a number of highly conserved functional domains, including an RCC1 domain15–17
, two PHR repeats18
, a Myc-binding domain16
and a C-terminal RING-H2 finger E3 ubiquitin ligase domain9,19
. Studies in worms, flies and mice have found that the PHR E3 ubiquitin ligases associate with a highly conserved F-box protein—FSN-1 in worm, Fsn in fly or Fbox45 in mouse—and together they function as a SCF-like E3 ubiquitin ligase complex to regulate neural development20–22
. An important downstream target of the ubiquitin ligase complex is the MAP kinase kinase kinase (MAPKKK) Wallenda (Wnd), which activates a MAP kinase cascade to control synaptogenesis23,24
. Although considerable progress has been made in understanding the PHR-associated ligase complex and its downstream signaling cascade, very little is known about how PHR proteins are regulated. Autophagy can negatively regulate the abundance of Hiw protein25
in fly, but it is unclear how autophagy, a general protein-degradation pathway, can be controlled to modulate Hiw protein levels during synaptic development. Identifying Hiw cofactors that regulate Hiw activity and abundance is necessary to determine the mechanisms by which hiw
functions are controlled.
We identified one such Hiw cofactor, Drosophila melanogaster
Rae1. Rae1 encodes a 346 amino-acid protein that belongs to an evolutionary conserved WD40 repeat protein family. Three major functions of Rae1 have been reported in different organisms. First, Rae1 associates with microtubules in the cytoplasm and regulates the organization of the cytoskeletal network during mitosis26
. Second, Rae1 binds to Nup98 to facilitate the transportation of poly-A RNA from the nucleus to the cytosol27
. Third, Rae1 is an anaphase-promoting complex (APC)-associated protein that inhibits the targeting of Securin by the APC ubiquitin ligase complex, thereby regulating entry into anaphase during mitosis28
. In cultured Drosophila
SL2 cells, knockdown of Rae1 by RNA interference induces arrest in G1 phase, but results in no mRNA transport defects, suggesting that Rae1 is involved in cell cycle regulation, but not in mRNA export, in these cells29
. Although Rae1 binds microtubules in cultured mammalian neurons30
, the function of Rae1 in the nervous system has not yet been studied. We found that Rae1 is a binding partner and positive regulator of Hiw. Rae1 and Hiw physically and genetically interacted and worked together to restrain synaptic terminal growth. These results indicate that the function of Rae1 in the Hiw E3 ubiquitin ligase complex is to prevent the autophagy-mediated degradation of Hiw protein in post-mitotic neurons. Our findings, and those of others (B. Grill, L. Chen, E.D. Tulgren, S.T. Baker, W. Bienvenut et al.
, personal communication), suggest an evolutionarily conserved role for Rae1 in the regulation of neural development.