Yohei Ohashi

The A-Type Cyclin CYCA2;3 Is a Key Regulator of Ploidy Levels in Arabidopsis Endoreduplication

Plant cells frequently undergo endoreduplication, a process in which chromosomal DNA is successively duplicated in the absence of mitosis. It has been proposed that endoreduplication is regulated at its entry by mitotic cyclin-dependent kinase activity. However, the regulatory mechanisms for its termination remain unclear, although plants tightly control the ploidy level in each cell type. In the process of searching for regulatory factors of endoreduplication, the promoter of an Arabidopsis thaliana cyclin A gene, CYCA2;3, was revealed to be active in developing trichomes during the termination period of endoreduplication as well as in proliferating tissues. Taking advantage of the situation that plants encode highly redundant cyclin A genes, we were able to perform functional dissection of CYCA2;3 using null mutant alleles. Null mutations of CYCA2;3 semidominantly promoted endocycles and increased the ploidy levels achieved in mature organs, but they did not significantly affect the proportion of cells that underwent endoreduplication. Consistent with this result, expression of the CYCA2;3–green fluorescent protein fusion protein restrained endocycles in a dose-dependent manner. Moreover, a mutation in the destruction box of CYCA2;3 stabilized the fusion protein in the nuclei and enhanced the restraint. We conclude that CYCA2;3 negatively regulates endocycles and acts as a key regulator of ploidy levels in Arabidopsis endoreduplication.

Membrane delivery to the yeast autophagosome from the Golgi-endosomal system.

The integral membrane protein Atg9 is delivered to the autophagosome in yeast and mammalian cells. We find that Atg9 does not originate from mitochondria and cannot reach the autophagosome directly from the ER. Instead, pairwise combinations of mutations in Golgi-endosomal traffic components cause defects in Atg9 delivery during starvation.

Structure and flexibility of the endosomal Vps34 complex reveals the basis of its function on membranes.

Opening up Vps34 protein complexes During intracellular membrane trafficking, large protein complexes regulate and adapt the activity of signal transducer enzymes such as the class III phosphatidylinositol 3-kinase Vps34. These large enzyme complexes are present in all eukaryotic cells, having widespread importance in neurodegeneration, aging, and cancer; however, a structural understanding has been lacking. Rostislavleva et al. provide atomic-resolution insights into the structures of the Vps34-containing protein complexes required for autophagy, endocytic sorting, and cytokinesis. The V-shaped complexes can undergo opening motions, which allows them to adapt to and phosphorylate membranes. Science, this issue p. 10.1126/science.aac7365 An atomic-resolution analysis provides insight into protein complexes required for autophagy, endocytic sorting, and cytokinesis. INTRODUCTION The lipid kinase Vps34/PIK3C3 phosphorylates phosphatidylinositol to yield phosphatidylinositol 3-phosphate (PI3P). Vps34 is important for processes that sort cargo to lysosomes, including phagocytosis, endocytic traffic, autophagy, and cytosol-to-vacuole transport. In mammalian cells, the enzyme also has roles in cytokinesis, signaling, recycling, and lysosomal tubulation. Vps34 is present in multiple complexes. Complex I functions in autophagy and contains Vps34, Vps15 (p150/PIK3R4 in mammals), Vps30/Atg6 (Beclin 1), and Atg14 (ATG14L). Complex II takes part in endocytic sorting (as well as autophagy and cytokinesis in mammalian cells) and contains the same subunits as complex I, except that it has Vps38 (UVRAG) instead of Atg14. These complexes are differentially regulated in stress responses. In autophagy, PI3P emerges on small tubular or vesicular structures associated with nascent autophagosomes. RATIONALE One of the most compelling questions is how the Vps34-containing complexes are organized and to what extent their intrinsic properties contribute to their differential activities in cells. To understand the mechanisms by which these complexes impart differential activities to Vps34, we sought to determine the structure of complex II and to characterize activities of Vps34 complexes on small and large vesicles. Because the complex resisted crystallization attempts, we screened 15 different nanobodies against the complex, and one of them enabled crystallization. RESULTS We obtained a 4.4 Å crystal structure of yeast complex II. The structure has a Y-shaped organization with the Vps15 and Vps34 subunits intertwining in one arm so that the Vps15 kinase domain interacts with the lipid-binding region of the Vps34 kinase domain. The other arm has a parallel Vps30/Vps38 heterodimer. This indicates that the complex might assemble by Vps15/Vps34 associating with Vps30/Vps38. This assembly path is consistent with in vitro reconstitution of complex II and suggests how the abundance of various Vps34-containing complexes might be dynamically controlled. The Vps34 C2 domain is the keystone to the organization of the complexes, and several structural elaborations of the domain that facilitate its interaction with all complex II subunits are essential to the cellular role of Vps34. We used hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify localized changes in all four complex II subunits upon membrane binding. We identified a loop in Vps30 (referred to as the “aromatic finger”) that interacts directly with lipid membranes. Our assays showed that complexes I and II had similar activities on small vesicles (100 nm). In contrast, only complex II was active on giant unilamellar vesicles (GUVs) (2 to 20 μm). This activity was completely abolished by mutation of the aromatic finger. CONCLUSION The structure, HDX-MS, and functional data allowed us to devise a model of how Vps34 complexes adapt to membranes. The tips of both arms of complex II work together on membranes. The Vps30 aromatic finger in one arm is important for the efficient catalytic activity of the other arm. The conformational changes that we detected may allow the arms to open to accommodate low-curvature membranes such as GUVs and endosomes. Most of the interactions observed in the complex II structure are likely to be detected in complex I as well. The restriction of complex I activity in autophagy to membrane structures smaller than 100 nm may be related to the inactivity of complex I on GUVs in vitro. Structure of complex II and its activity on GUVs. In the Y-shaped complex II, the Vps30/Vps38 pair in one arm brackets the Vps15/Vps34 pair in the other arm. Tips of both arms bind membranes. Only wild-type complex II forms PI3P on GUVs; in contrast, complex I and the complex II aromatic finger mutant are inactive. PI3P is detected by a sensor protein (red) binding to GUVs (green). Both complexes I and II have similar activities on small vesicles. Phosphatidylinositol 3-kinase Vps34 complexes regulate intracellular membrane trafficking in endocytic sorting, cytokinesis, and autophagy. We present the 4.4 angstrom crystal structure of the 385-kilodalton endosomal complex II (PIK3C3-CII), consisting of Vps34, Vps15 (p150), Vps30/Atg6 (Beclin 1), and Vps38 (UVRAG). The subunits form a Y-shaped complex, centered on the Vps34 C2 domain. Vps34 and Vps15 intertwine in one arm, where the Vps15 kinase domain engages the Vps34 activation loop to regulate its activity. Vps30 and Vps38 form the other arm that brackets the Vps15/Vps34 heterodimer, suggesting a path for complex assembly. We used hydrogen-deuterium exchange mass spectrometry (HDX-MS) to reveal conformational changes accompanying membrane binding and identify a Vps30 loop that is critical for the ability of complex II to phosphorylate giant liposomes on which complex I is inactive.

GLABRA2 Directly Suppresses Basic Helix-Loop-Helix Transcription Factor Genes with Diverse Functions in Root Hair Development

GLABRA2 targets and suppresses five basic helix-loop-helix transcription factor genes with diverse functions in root hair development during root hair pattern formation of Arabidopsis thaliana. The Arabidopsis thaliana GLABRA2 (GL2) gene encodes a transcription factor involved in the cell differentiation of various epidermal tissues. During root hair pattern formation, GL2 suppresses root hair development in non-hair cells, acting as a node between the gene regulatory networks for cell fate determination and cell differentiation. Despite the importance of GL2 function, its molecular basis remains obscure because the GL2 target genes leading to the network for cell differentiation are unknown. We identified five basic helix-loop-helix (bHLH) transcription factor genes (ROOT HAIR DEFECTIVE6 [RHD6], RHD6-LIKE1 [RSL1], RSL2, Lj-RHL1-LIKE1 [LRL1], and LRL2) as GL2 direct targets using transcriptional and posttranslational induction systems. Chromatin immunoprecipitation analysis confirmed GL2 binding to upstream regions of these genes in planta. Reporter gene analyses showed that these genes are expressed in various stages of root hair development and are suppressed by GL2 in non-hair cells. GL2 promoter-driven GFP fusions of LRL1 and LRL2, but not those of the other bHLH proteins, conferred root hair development on non-hair cells. These results indicate that GL2 directly suppresses bHLH genes with diverse functions in root hair development.

Characterization of Atg38 and NRBF2, a fifth subunit of the autophagic Vps34/PIK3C3 complex

ABSTRACT The phosphatidylinositol 3-kinase Vps34 is part of several protein complexes. The structural organization of heterotetrameric complexes is starting to emerge, but little is known about organization of additional accessory subunits that interact with these assemblies. Combining hydrogen-deuterium exchange mass spectrometry (HDX-MS), X-ray crystallography and electron microscopy (EM), we have characterized Atg38 and its human ortholog NRBF2, accessory components of complex I consisting of Vps15-Vps34-Vps30/Atg6-Atg14 (yeast) and PIK3R4/VPS15-PIK3C3/VPS34-BECN1/Beclin 1-ATG14 (human). HDX-MS shows that Atg38 binds the Vps30-Atg14 subcomplex of complex I, using mainly its N-terminal MIT domain and bridges the coiled-coil I regions of Atg14 and Vps30 in the base of complex I. The Atg38 C-terminal domain is important for localization to the phagophore assembly site (PAS) and homodimerization. Our 2.2 Å resolution crystal structure of the Atg38 C-terminal homodimerization domain shows 2 segments of α-helices assembling into a mushroom-like asymmetric homodimer with a 4-helix cap and a parallel coiled-coil stalk. One Atg38 homodimer engages a single complex I. This is in sharp contrast to human NRBF2, which also forms a homodimer, but this homodimer can bridge 2 complex I assemblies.