Dongyan Tan

The EM structure of the TRAPPIII complex leads to the identification of a requirement for COPII vesicles on the macroautophagy pathway

Significance Rab GTPases are critical determinants of membrane identity in eukaryotic cells. Typically, each GTPase is activated by a single guanine nucleotide exchange factor (GEF), but the Ypt1/Rab1 GTPase is activated by three related GEFs, transport protein particle (TRAPP) I, TRAPPII, and TRAPPIII, that function in distinct trafficking pathways. TRAPPIII is recruited to the phagophore assembly site when macroautophagy is induced. Here, we present the single-particle electron microscopy structure of TRAPPIII and evidence that TRAPPIII binds the coat protein complex (COP) II coat. We also show that the ER-Golgi fusion machinery that is present on COPII vesicles is needed for macroautophagy. Our results imply that TRAPPIII recruits COPII vesicles to the PAS and that COPII vesicles may provide one of the membrane sources used in autophagosome formation. The transport protein particle (TRAPP) III complex, comprising the TRAPPI complex and additional subunit Trs85, is an autophagy-specific guanine nucleotide exchange factor for the Rab GTPase Ypt1 that is recruited to the phagophore assembly site when macroautophagy is induced. We present the single-particle electron microscopy structure of TRAPPIII, which reveals that the dome-shaped Trs85 subunit associates primarily with the Trs20 subunit of TRAPPI. We further demonstrate that TRAPPIII binds the coat protein complex (COP) II coat subunit Sec23. The COPII coat facilitates the budding and targeting of ER-derived vesicles with their acceptor compartment. We provide evidence that COPII-coated vesicles and the ER-Golgi fusion machinery are needed for macroautophagy. Our results imply that TRAPPIII binds to COPII vesicles at the phagophore assembly site and that COPII vesicles may provide one of the membrane sources used in autophagosome formation. These events are conserved in yeast to mammals.

Kinesin-13s form rings around microtubules

Kinesin is a superfamily of motor proteins that uses the energy of adenosine triphosphate hydrolysis to move and generate force along microtubules. A notable exception to this general description is found in the kinesin-13 family that actively depolymerizes microtubules rather than actively moving along them. This depolymerization activity is important in mitosis during chromosome segregation. It is still not fully clear by which mechanism kinesin-13s depolymerize microtubules. To address this issue, we used electron microscopy to investigate the interaction of kinesin-13s with microtubules. Surprisingly, we found that proteins of the kinesin-13 family form rings and spirals around microtubules. This is the first report of this type of oligomeric structure for any kinesin protein. These rings may allow kinesin-13s to stay at the ends of microtubules during depolymerization.

Structural Model for Tubulin Recognition and Deformation by Kinesin-13 Microtubule Depolymerases

To elucidate the structural basis of the mechanism of microtubule depolymerization by kinesin-13s, we analyzed complexes of tubulin and the Drosophila melanogaster kinesin-13 KLP10A by electron microscopy (EM) and fluorescence polarization microscopy. We report a nanometer-resolution (1.1 nm) cryo-EM three-dimensional structure of the KLP10A head domain (KLP10AHD) bound to curved tubulin. We found that binding of KLP10AHD induces a distinct tubulin configuration with displacement (shear) between tubulin subunits in addition to curvature. In this configuration, the kinesin-binding site differs from that in straight tubulin, providing an explanation for the distinct interaction modes of kinesin-13s with the microtubule lattice or its ends. The KLP10AHD-tubulin interface comprises three areas of interaction, suggesting a crossbow-type tubulin-bending mechanism. These areas include the kinesin-13 family conserved KVD residues, and as predicted from the crossbow model, mutating these residues changes the orientation and mobility of KLP10AHDs interacting with the microtubule.

Structure of the Kinesin13-Microtubule Ring Complex

To investigate the mechanism of kinesin13-induced microtubule depolymerization, we have calculated a three-dimensional (3D) map of the kinesin13-microtubule ring complex, using cryo-electron microscopy (cryo-EM) and image analysis. An atomic model of the complex was produced by docking the crystal structures of tubulin and a kinesin13 motor domain (MD) into the 3D map. The model reveals a snapshot of the depolymerization mechanism by providing a 3D view of the complex formed between the kinesin13 MD and a curved tubulin protofilament (pf). It suggests that contacts mediated by kinesin13 class-specific residues in the putative microtubule-binding site stabilize intra-dimer tubulin curvature. In addition, a tubulin-binding site on the kinesin13 MD was identified. Mutations at this class-conserved site selectively disrupt the formation of microtubule-associated ring complexes.

Structural analyses of the chromatin remodelling enzymes INO80-C and SWR-C

INO80-C and SWR-C are conserved members of a subfamily of ATP-dependent chromatin remodeling enzymes that function in transcription and genome-maintenance pathways. A crucial role for these enzymes is to control chromosomal distribution of the H2A.Z histone variant. Here we use electron microscopy (EM) and two-dimensional (2D) class averaging to demonstrate that these remodeling enzymes have similar overall architectures. Each enzyme is characterized by a dynamic ‘tail’ domain and a compact ‘head’ that contains Rvb1/Rvb2 subunits organized as hexameric rings. EM class averages and mass spectrometry support the existence of single heterohexameric rings in both SWR-C and INO80-C. EM studies define the position of the Arp8/Arp4/Act1 module within INO80-C, and we find that this module enhances nucleosome binding affinity but is largely dispensable for remodeling activities. In contrast, the Ies6/Arp5 module is essential for INO80-C remodeling, and furthermore this module controls conformational changes that may couple nucleosome binding to remodeling.

Unique double-ring structure of the peroxisomal Pex1/Pex6 ATPase complex revealed by cryo-electron microscopy

Significance Pex1 and Pex6 are members of the AAA family of ATPases, which contain two ATPase domains in a single polypeptide chain and form hexameric double rings. These two Pex proteins are involved in the biogenesis of peroxisomes, and mutations in them frequently cause diseases. Here, we determined structures of the Pex1/Pex6 complex by cryo-electron microscopy. Novel computational modeling methods allowed placement of Pex1/Pex6 domains into subnanometer density maps. Our results show that the peroxisomal Pex1/Pex6 ATPases form a unique double-ring structure in which the two proteins alternate around the ring. Our data shed light on the mechanism and function of this ATPase and suggest a role in peroxisomal protein import similar to that of p97 in ER-associated protein degradation. Members of the AAA family of ATPases assemble into hexameric double rings and perform vital functions, yet their molecular mechanisms remain poorly understood. Here, we report structures of the Pex1/Pex6 complex; mutations in these proteins frequently cause peroxisomal diseases. The structures were determined in the presence of different nucleotides by cryo-electron microscopy. Models were generated using a computational approach that combines Monte Carlo placement of structurally homologous domains into density maps with energy minimization and refinement protocols. Pex1 and Pex6 alternate in an unprecedented hexameric double ring. Each protein has two N-terminal domains, N1 and N2, structurally related to the single N domains in p97 and N-ethylmaleimide sensitive factor (NSF); N1 of Pex1 is mobile, but the others are packed against the double ring. The N-terminal ATPase domains are inactive, forming a symmetric D1 ring, whereas the C-terminal domains are active, likely in different nucleotide states, and form an asymmetric D2 ring. These results suggest how subunit activity is coordinated and indicate striking similarities between Pex1/Pex6 and p97, supporting the hypothesis that the Pex1/Pex6 complex has a role in peroxisomal protein import analogous to p97 in ER-associated protein degradation.

Motor domain phosphorylation and regulation of the Drosophila kinesin 13, KLP10A

Microtubule (MT)-destabilizing kinesin 13s perform fundamental roles throughout the cell cycle. In this study, we show that the Drosophila melanogaster kinesin 13, KLP10A, is phosphorylated in vivo at a conserved serine (S573) positioned within the α-helix 5 of the motor domain. In vitro, a phosphomimic KLP10A S573E mutant displays a reduced capacity to depolymerize MTs but normal affinity for the MT lattice. In cells, replacement of endogenous KLP10A with KLP10A S573E dampens MT plus end dynamics throughout the cell cycle, whereas a nonphosphorylatable S573A mutant apparently enhances activity during mitosis. Electron microscopy suggests that KLP10A S573 phosphorylation alters its association with the MT lattice, whereas molecular dynamics simulations reveal how KLP10A phosphorylation can alter the kinesin–MT interface without changing important structural features within the motor’s core. Finally, we identify casein kinase 1α as a possible candidate for KLP10A phosphorylation. We propose a model in which phosphorylation of the KLP10A motor domain provides a regulatory switch controlling the time and place of MT depolymerization.

The Drosophila Kinesin-13, KLP59D, Impacts Pacman and Flux-based Chromosome Movement

Chromosome movements are linked to the active depolymerization of spindle microtubule (MT) ends. Here we identify the kinesin-13 family member, KLP59D, as a novel and uniquely important regulator of spindle MT dynamics and chromosome motility in Drosophila somatic cells. During prometaphase and metaphase, depletion of KLP59D, which targets to centrosomes and outer kinetochores, suppresses the depolymerization of spindle pole-associated MT minus ends, thereby inhibiting poleward tubulin Flux. Subsequently, during anaphase, loss of KLP59D strongly attenuates chromatid-to-pole motion by suppressing the depolymerization of both minus and plus ends of kinetochore-associated MTs. The mechanism of KLP59Ds impact on spindle MT plus and minus ends appears to differ. Our data support a model in which KLP59D directly depolymerizes kinetochore-associated plus ends during anaphase, but influences minus ends indirectly by localizing the pole-associated MT depolymerase KLP10A. Finally, electron microscopy indicates that, unlike the other Drosophila kinesin-13s, KLP59D is largely incapable of oligomerizing into MT-associated rings in vitro, suggesting that such structures are not a requisite feature of kinetochore-based MT disassembly and chromosome movements.

UtpA and UtpB chaperone nascent pre-ribosomal RNA and U3 snoRNA to initiate eukaryotic ribosome assembly

Early eukaryotic ribosome biogenesis involves large multi-protein complexes, which co-transcriptionally associate with pre-ribosomal RNA to form the small subunit processome. The precise mechanisms by which two of the largest multi-protein complexes—UtpA and UtpB—interact with nascent pre-ribosomal RNA are poorly understood. Here, we combined biochemical and structural biology approaches with ensembles of RNA–protein cross-linking data to elucidate the essential functions of both complexes. We show that UtpA contains a large composite RNA-binding site and captures the 5′ end of pre-ribosomal RNA. UtpB forms an extended structure that binds early pre-ribosomal intermediates in close proximity to architectural sites such as an RNA duplex formed by the 5′ ETS and U3 snoRNA as well as the 3′ boundary of the 18S rRNA. Both complexes therefore act as vital RNA chaperones to initiate eukaryotic ribosome assembly.

A requirement for ER-derived COPII vesicles in phagophore initiation

A major unanswered question in the field of autophagy is how the double-membrane phagophore is formed. As this membrane expands, it engulfs proteins and organelles that are destined for degradation and then seals to form an autophagosome. A growing consensus in the field is that a subdomain of the ER initiates formation of the phagophore. We show that ER-derived COPII-coated vesicles, which bud from a specialized domain of the ER called the ER exit site (ERES), are a source of this membrane. This finding will now pave the way for a biochemical description of the early steps of phagophore initiation.