Guangpu Li

Structure of the APPL1 BAR-PH domain and characterization of its interaction with Rab5.

APPL1 is an effector of the small GTPase Rab5. Together, they mediate a signal transduction pathway initiated by ligand binding to cell surface receptors. Interaction with Rab5 is confined to the amino (N)‐terminal region of APPL1. We report the crystal structures of human APPL1 N‐terminal BAR‐PH domain motif. The BAR and PH domains, together with a novel linker helix, form an integrated, crescent‐shaped, symmetrical dimer. This BAR–PH interaction is likely conserved in the class of BAR‐PH containing proteins. Biochemical analyses indicate two independent Rab‐binding sites located at the opposite ends of the dimer, where the PH domain directly interacts with Rab5 and Rab21. Besides structurally supporting the PH domain, the BAR domain also contributes to Rab binding through a small surface region in the vicinity of the PH domain. In stark contrast to the helix‐dominated, Rab‐binding domains previously reported, APPL1 PH domain employs β‐strands to interact with Rab5. On the Rab5 side, both switch regions are involved in the interaction. Thus we identified a new binding mode between PH domains and small GTPases.

Structural basis of Rab5-Rabaptin5 interaction in endocytosis

Rab5 is a small GTPase that regulates early endosome fusion. We present here the crystal structure of the Rab5 GTPase domain in complex with a GTP analog and the C-terminal domain of effector Rabaptin5. The proteins form a dyad-symmetric Rab5–Rabaptin52–Rab5 ternary complex with a parallel coiled-coil Rabaptin5 homodimer in the middle. Two Rab5 molecules bind independently to the Rabaptin5 dimer using their switch and interswitch regions. The binding does not involve the Rab complementarity-determining regions. We also present the crystal structures of two distinct forms of GDP–Rab5 complexes, both of which are incompatible with Rabaptin5 binding. One has a dislocated and disordered switch I but a virtually intact switch II, whereas the other has its β-sheet and both switch regions reorganized. Biochemical and functional analyses show that the crystallographically observed Rab5–Rabaptin5 complex also exists in solution, and disruption of this complex by mutation abrogates endosome fusion.

Rac1 is required for pathogenicity and Chm1-dependent conidiogenesis in rice fungal pathogen Magnaporthe grisea.

Rac1 is a small GTPase involved in actin cytoskeleton organization and polarized cell growth in many organisms. In this study, we investigate the biological function of MgRac1, a Rac1 homolog in Magnaporthe grisea. The Mgrac1 deletion mutants are defective in conidial production. Among the few conidia generated, they are malformed and defective in appressorial formation and consequently lose pathogenicity. Genetic complementation with native MgRac1 fully recovers all these defective phenotypes. Consistently, expression of a dominant negative allele of MgRac1 exhibits the same defect as the deletion mutants, while expression of a constitutively active allele of MgRac1 can induce abnormally large conidia with defects in infection-related growth. Furthermore, we show the interactions between MgRac1 and its effectors, including the PAK kinase Chm1 and NADPH oxidases (Nox1 and Nox2), by the yeast two-hybrid assay. While the Nox proteins are important for pathogenicity, the MgRac1-Chm1 interaction is responsible for conidiogenesis. A constitutively active chm1 mutant, in which the Rac1-binding PBD domain is removed, fully restores conidiation of the Mgrac1 deletion mutants, but these conidia do not develop appressoria normally and are not pathogenic to rice plants. Our data suggest that the MgRac1-Chm1 pathway is responsible for conidiogenesis, but additional pathways, including the Nox pathway, are necessary for appressorial formation and pathogenicity.

Catalytic Domain of the p120 Ras GAP Binds to Rab5 and Stimulates Its GTPase Activity

Ras is a master GTPase switch controlling multiple signal transduction cascades in the regulation of cell proliferation and differentiation. Rab5 is a local GTPase switch that is localized on early endosomes and controls early endosome fusion. This study demonstrates that the catalytic domain of p120 GTPase-activating protein (GAP), a well known Ras GAP, is able to interact physically with Rab5 and stimulate its GTPase activity. This GAP activity toward Rab5, however, cannot be extended to other Rab GTPases such as Rab3, Rab4, and Rab6, indicating that it is not a generic GAP for the Rab family of GTPases that regulate intracellular membrane fusion during endocytosis and exocytosis. The findings indicate a level of structural similarity between Ras and Rab5 unexpected from their primary sequences. They also suggest a possible signal transduction regulation of the Rab5-dependent endosome fusion via the Ras GAP.

Structure of human lanthionine synthetase C-like protein 1 and its interaction with Eps8 and glutathione.

Eukaryotic lanthionine synthetase C-like protein 1 (LanCL1) is homologous to prokaryotic lanthionine cyclases, yet its biochemical functions remain elusive. We report the crystal structures of human LanCL1, both free of and complexed with glutathione, revealing glutathione binding to a zinc ion at the putative active site formed by conserved GxxG motifs. We also demonstrate by in vitro affinity analysis that LanCL1 binds specifically to the SH3 domain of a signaling protein, Eps8. Importantly, expression of LanCL1 mutants defective in Eps8 interaction inhibits nerve growth factor (NGF)-induced neurite outgrowth, providing evidence for the biological significance of this novel interaction in cellular signaling and differentiation.

Cell cycle‐dependent ubiquitylation and destruction of NDE1 by CDK5‐FBW7 regulates ciliary length

Primary cilia start forming within the G1 phase of the cell cycle and continue to grow as cells exit the cell cycle (G0). They start resorbing when cells re‐enter the cell cycle (S phase) and are practically invisible in mitosis. The mechanisms by which cilium biogenesis and disassembly are coupled to the cell cycle are complex and not well understood. We previously identified the centrosomal phosphoprotein NDE1 as a negative regulator of ciliary length and showed that its levels inversely correlate with ciliogenesis. Here, we identify the tumor suppressor FBW7 (also known as FBXW7, CDC4, AGO, or SEL‐10) as the E3 ligase that mediates the destruction of NDE1 upon entry into G1. CDK5, a kinase active in G1/G0, primes NDE1 for FBW7‐mediated recognition. Cells depleted of FBW7 or CDK5 show enhanced levels of NDE1 and a reduction in ciliary length, which is corrected in cells depleted of both FBW7 or CDK5 and NDE1. These data show that cell cycle‐dependent mechanisms can control ciliary length through a CDK5‐FBW7‐NDE1 pathway.

Biogenesis and Function of the NGF/TrkA Signaling Endosome

Target-derived neurotrophin nerve growth factor (NGF) and its receptor TrkA are well known for retrograde signaling to promote survival and innervation of sympathetic and sensory neurons. In recent years, the signaling endosome model has been used to describe the sustained NGF/TrkA retrograde signaling as a process of endocytosis and retrograde transport of NGF/TrkA-containing endosomes from the axon terminal to the cell body for activation of NGF-inducible gene expression responsible for neuronal survival and development. Here, we review the biogenesis and function of NGF, TrkA, and the signaling endosome and discuss possible roles of Rab GTPases in the biogenesis and trafficking of signaling endosomes.

Rab22 controls NGF signaling and neurite outgrowth in PC12 cells

Rab22 is a small GTPase that is localized on early endosomes and regulates early endosomal sorting. This study reports that Rab22 promotes NGF signaling–dependent neurite outgrowth and gene expression in PC12 cells by sorting NGF and the activated/phosphorylated receptor (pTrkA) into signaling endosomes to sustain signal transduction in the cell.

Crystal structure of human GGA1 GAT domain complexed with the GAT-binding domain of Rabaptin5

GGA proteins coordinate the intracellular trafficking of clathrin‐coated vesicles through their interaction with several other proteins. The GAT domain of GGA proteins interacts with ARF, ubiquitin, and Rabaptin5. The GGA–Rabaptin5 interaction is believed to function in the fusion of trans‐Golgi‐derived vesicles to endosomes. We determined the crystal structure of a human GGA1 GAT domain fragment in complex with the Rabaptin5 GAT‐binding domain. In this structure, the Rabaptin5 domain is a 90‐residue‐long helix. At the N‐terminal end, it forms a parallel coiled‐coil homodimer, which binds one GAT domain of GGA1. In the C‐terminal region, it further assembles into a four‐helix bundle tetramer. The Rabaptin5‐binding motif of the GGA1 GAT domain consists of a three‐helix bundle. Thus, the binding between Rabaptin5 and GGA1 GAT domain is based on a helix bundle–helix bundle interaction. The current structural observation is consistent with previously reported mutagenesis data, and its biological relevance is further confirmed by new mutagenesis studies and affinity analysis. The four‐helix bundle structure of Rabaptin5 suggests a functional role in tethering organelles.

Retromer Is Essential for Autophagy-Dependent Plant Infection by the Rice Blast Fungus

The retromer mediates protein trafficking through recycling cargo from endosomes to the trans-Golgi network in eukaryotes. However, the role of such trafficking events during pathogen-host interaction remains unclear. Here, we report that the cargo-recognition complex (MoVps35, MoVps26 and MoVps29) of the retromer is essential for appressorium-mediated host penetration by Magnaporthe oryzae, the causal pathogen of the blast disease in rice. Loss of retromer function blocked glycogen distribution and turnover of lipid bodies, delayed nuclear degeneration and reduced turgor during appressorial development. Cytological observation revealed dynamic MoVps35-GFP foci co-localized with autophagy-related protein RFP-MoAtg8 at the periphery of autolysosomes. Furthermore, RFP-MoAtg8 interacted with MoVps35-GFP in vivo, RFP-MoAtg8 was mislocalized to the vacuole and failed to recycle from the autolysosome in the absence of the retromer function, leading to impaired biogenesis of autophagosomes. We therefore conclude that retromer is essential for autophagy-dependent plant infection by the rice blast fungus.