Jia, Da

Pontocerebellar hypoplasia (PCH) is a group of rare neurodevelopmental disorders with limited diagnostic and therapeutic options. Mutations in WDR11, a subunit of the FAM91A1 complex, have been found in patients with PCH-like symptoms; however, definitive evidence that the mutations are causal is still lacking. Here, we show that depletion of FAM91A1 results in developmental defects in zebrafish similar to that of TBC1D23, an established PCH gene. FAM91A1 and TBC1D23 directly interact with each other and cooperate to regulate endosome-to-Golgi trafficking of KIAA0319L, a protein known to regulate axonal growth. Crystal structure of the FAM91A1–TBC1D23 complex reveals that TBC1D23 binds to a conserved surface on FAM91A1 by assuming a Z-shaped conformation. More importantly, the interaction between FAM91A1 and TBC1D23 can be used to predict the risk of certain TBC1D23-associated mutations to PCH. Collectively, our study provides a molecular basis for the interaction between TBC1D23 and FAM91A1 and suggests that disrupted endosomal trafficking underlies multiple PCH subtypes.

Understanding of the evolution of metazoans from their unicellular ancestors is a fundamental question in biology. In contrast to fungi which utilize the Mon1–Ccz1 dimeric complex to activate the small GTPase RAB7A, metazoans rely on the Mon1–Ccz1–RMC1 trimeric complex. Here, we report a near-atomic resolution cryogenic-electron microscopy structure of the Drosophila Mon1–Ccz1–RMC1 complex. RMC1 acts as a scaffolding subunit and binds to both Mon1 and Ccz1 on the surface opposite to the RAB7A-binding site, with many of the RMC1-contacting residues from Mon1 and Ccz1 unique to metazoans, explaining the binding specificity. Significantly, the assembly of RMC1 with Mon1–Ccz1 is required for cellular RAB7A activation, autophagic functions and organismal development in zebrafish. Our studies offer a molecular explanation for the different degree of subunit conservation across species, and provide an excellent example of how metazoan-specific proteins take over existing functions in unicellular organisms.

Significance The sorting nexin proteins are critical for endosomal trafficking and signaling. SNX17 and SNX27 are two SNX proteins displaying high similarity in domain structure, including a common FERM domain; however, they mediate distinct endocytic recycling pathways. We now solve this mystery by demonstrating that the FERM domains of SNX17 and SNX27 have different functions. Whereas the FERM domain of SNX17 recognizes the NPxY/NxxY motif, the same domain in SNX27 binds to a novel “DLF” motif within the N termini of SNX1/2. The interaction between SNX27 and SNX1/2 not only helps efficient retrieval of multiple cargoes but also promotes endosomal recruitment of SNX27. We further demonstrate that the SNX27–SNX1/2 interaction is crucial to neuronal growth and brain development in zebrafish.

Endocytosed proteins can be delivered to lysosomes for degradation or recycled to either the trans-Golgi network or the plasma membrane. It remains poorly understood how the recycling versus degradation of cargoes is determined. Here, we show that multiple extracellular stimuli, including starvation, LPS, IL-6, and EGF treatment, can strongly inhibit endocytic recycling of multiple cargoes through the activation of MAPK11/14. The stress-induced kinases in turn directly phosphorylate SNX27, a key regulator of endocytic recycling, at serine 51 (Ser51). Phosphorylation of SNX27 at Ser51 alters the conformation of its cargo-binding pocket and decreases the interaction between SNX27 and cargo proteins, thereby inhibiting endocytic recycling. Our study indicates that endocytic recycling is highly dynamic and can crosstalk with cellular stress–signaling pathways. Suppression of endocytic recycling and enhancement of receptor lysosomal degradation serve as new mechanisms for cells to cope with stress and save energy.

Pontocerebellar hypoplasia (PCH) is a group of neurological disorders that affect the development of the brain, in particular, the pons and cerebellum. Homozygous mutations of TBC1D23 have been found recently to lead to PCH; however, the underlying molecular mechanisms remain unclear. Here, we show that the crystal structure of the TBC1D23 C-terminal domain adopts a Pleckstrin homology domain fold and selectively binds to phosphoinositides, in particular, PtdIns(4)P, through one surface while binding FAM21 via the opposite surface. Mutation of key residues of TBC1D23 or FAM21 selectively disrupts the endosomal vesicular trafficking toward the Trans-Golgi Network. Finally, using the zebrafish model, we show that PCH patient-derived mutants, impacting either phosphoinositide binding or FAM21 binding, lead to abnormal neuronal growth and brain development. Taken together, our data provide a molecular basis for the interaction between TBC1D23 and FAM21, and suggest a plausible role for PtdIns(4)P in the TBC1D23-mediating endosome-to-TGN trafficking pathway. Defects in this trafficking pathway are, at least partially, responsible for the pathogenesis of certain types of PCH.

Notch family members are transmembrane receptors that mediate essential developmental programs. Upon ligand binding, a proteolytic event releases the intracellular domain of Notch, which translocates to the nucleus to regulate gene transcription. In addition, Notch trafficking across the endolysosomal system is critical in its regulation. In this study we report that Notch recycling to the cell surface is dependent on the COMMD–CCDC22–CCDC93 (CCC) complex, a recently identified regulator of endosomal trafficking. Disruption in this system leads to intracellular accumulation of Notch2 and concomitant reduction in Notch signaling. Interestingly, among the 10 copper metabolism MURR1 domain containing (COMMD) family members that can associate with the CCC complex, only COMMD9 and its binding partner, COMMD5, have substantial effects on Notch. Furthermore, Commd9 deletion in mice leads to embryonic lethality and complex cardiovascular alterations that bear hallmarks of Notch deficiency. Altogether, these studies highlight that the CCC complex controls Notch activation by modulating its intracellular trafficking and demonstrate cargo-specific effects for members of the COMMD protein family.

Wiskott–Aldrich syndrome protein (WASPs) control actin dynamics in cellular processes, including cell motility, receptor-mediated endocytosis, bacterial invasion, and vesicular trafficking. We demonstrated that WASH, a recently identified WASP family protein, colocalizes on endosomal subdomains with the cargo-selective complex (CSC) of the retromer, where it regulates retrograde sorting from endosomes in an actin-dependent manner. However, the mechanism of WASH recruitment to these retromer-enriched endosomal subdomains is unclear. Here we show that a component of the WASH regulatory complex (SHRC), FAM21, which contains 21 copies of a novel L-F-[D/E]3-10-L-F motif, directly interacts with the retromer CSC protein VPS35. Endosomal localization of FAM21 is VPS35 dependent and relies on multivalency of FAM21 repeat elements. Using a combination of pull-down assays and isothermal calorimetry, we demonstrate that individual repeats can bind CSC, and binding affinity varies among different FAM21 repeats. A high-affinity repeat can be converted into a low-affinity one by mutation of a hydrophobic residue within the motif. These in vitro data mirror the localization of FAM21 to retromer-coated vesicles in cells. We propose that multivalency enables FAM21 to sense the density of retromer on membranes, allowing coordination of SHRC recruitment, and consequent actin polymerization, with retromer sorting domain organization/maturation.

We recently showed that the Wiskott–Aldrich syndrome protein (WASP) family member, WASH, localizes to endosomal subdomains and regulates endocytic vesicle scission in an Arp2/3-dependent manner. Mechanisms regulating WASH activity are unknown. Here we show that WASH functions in cells within a 500 kDa core complex containing Strumpellin, FAM21, KIAA1033 (SWIP), and CCDC53. Although recombinant WASH is constitutively active toward the Arp2/3 complex, the reconstituted core assembly is inhibited, suggesting that it functions in cells to regulate actin dynamics through WASH. FAM21 interacts directly with CAPZ and inhibits its actin-capping activity. Four of the five core components show distant (approximately 15% amino acid sequence identify) but significant structural homology to components of a complex that negatively regulates the WASP family member, WAVE. Moreover, biochemical and electron microscopic analyses show that the WASH and WAVE complexes are structurally similar. Thus, these two distantly related WASP family members are controlled by analogous structurally related mechanisms. Strumpellin is mutated in the human disease hereditary spastic paraplegia, and its link to WASH suggests that misregulation of actin dynamics on endosomes may play a role in this disorder.