Vrije Universiteit Brussel Structure and flexibility of the endosomal Vps34 complex reveals the basis of its function on membranes Rostislavleva, Ksenia; Soler, Nicolas; Ohashi, Yohei; Zhang, Lufei; Pardon, Els; Burke, John E; Masson, Glenn R; Johnson, Chris; Steyaert, Jan; Ktistakis, Nicholas T; Williams, Roger L Published in: Science DOI: 10.1126/science.aac7365 Publication date: 2015 License: Unspecified Document Version: Final published version Link to publication Citation for published version (APA): Rostislavleva, K., Soler, N., Ohashi, Y., Zhang, L., Pardon, E., Burke, J. E., ... Williams, R. L. (2015). Structure and flexibility of the endosomal Vps34 complex reveals the basis of its function on membranes. 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Oct. 2021 RESEARCH ◥ plex II and to characterize activities of Vps34 RESEARCH ARTICLE SUMMARY complexes on small and large vesicles. Be- cause the complex resisted crystallization attempts, we screened 15 different nanobodies STRUCTURAL BIOLOGY against the complex, and one of them enabled Structure and flexibility of the crystallization. RESULTS: We obtained a 4.4 Å crystal struc- endosomal Vps34 complex reveals the ture of yeast complex II. The structure has a Y-shaped organization with the Vps15 and Vps34 subunits intertwining in one arm so basis of its function on membranes that the Vps15 kinase domain interacts with ◥ the lipid-binding region ON OUR WEB SITE Ksenia Rostislavleva, Nicolas Soler, Yohei Ohashi, Lufei Zhang, Els Pardon, of the Vps34 kinase do- John E. Burke, Glenn R. Masson, Chris Johnson, Jan Steyaert, Read the full article main. The other arm has Nicholas T. Ktistakis, Roger L. Williams* at http://dx.doi. a parallel Vps30/Vps38 org/10.1126/ heterodimer. This indicates science.aac7365 that the complex might as- INTRODUCTION: The lipid kinase Vps34/ lian cells) and contains the same subunits as .................................................. PIK3C3 phosphorylates phosphatidylinositol complexI,exceptthatithasVps38(UVRAG) semble by Vps15/Vps34 to yield phosphatidylinositol 3-phosphate (PI3P). instead of Atg14. These complexes are differen- associating with Vps30/Vps38. This assembly Vps34 is important for processes that sort cargo tially regulated in stress responses. In autophagy, path is consistent with in vitro reconstitution to lysosomes, including phagocytosis, endocytic PI3P emerges on small tubular or vesicular struc- of complex II and suggests how the abundance traffic, autophagy, and cytosol-to-vacuole trans- tures associated with nascent autophagosomes. of various Vps34-containing complexes might port. In mammalian cells, the enzyme also has be dynamically controlled. The Vps34 C2 do- roles in cytokinesis, signaling, recycling, and RATIONALE: One of the most compelling main is the keystone to the organization of the lysosomal tubulation. questionsishowtheVps34-containingcom- complexes, and several structural elaborations on October 8, 2015 Vps34 is present in multiple complexes. Com- plexes are organized and to what extent their of the domain that facilitate its interaction plex I functions in autophagy and contains intrinsic properties contribute to their dif- with all complex II subunits are essential to Vps34, Vps15 (p150/PIK3R4 in mammals), ferential activities in cells. To understand the cellular role of Vps34. Vps30/Atg6 (Beclin 1), and Atg14 (ATG14L). the mechanisms by which these complexes We used hydrogen-deuterium exchange mass Complex II takes part in endocytic sorting (as impart differential activities to Vps34, we spectrometry (HDX-MS) to identify localized well as autophagy and cytokinesis in mamma- sought to determine the structure of com- 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 www.sciencemag.org 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 mm). This activity was completely abolished by mutation of the aromatic finger. CONCLUSION: The structure, HDX-MS, and Downloaded from functional data allowed us to devise a model of how Vps34 complexes adapt to membranes. ThetipsofbotharmsofcomplexIIwork together on membranes. The Vps30 aromatic finger in one arm is important for the ef- ficient 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 complexIIstructurearelikelytobedetected in complex I as well. The restriction of com- plex 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. The list of author affiliations is available in the full article online. Only wild-type complex II forms PI3P on GUVs; in contrast, complex I and the complex II aromatic *Corresponding author. E-mail: [email protected] finger mutant are inactive. PI3P is detected by a sensor protein (red) binding to GUVs (green). Both Cite this article as K. Rostislavleva et al., Science 350, complexes I and II have similar activities on small vesicles. aac7365 (2015). DOI: 10.1126/science.aac7365 178 9 OCTOBER 2015 • VOL 350 ISSUE 6257 sciencemag.org SCIENCE RESEARCH ◥ ing the structure was challenging at this resolution. RESEARCH ARTICLE Initial models for several domains of the complex derived from previous structures and distant homo- logs (16–18) were fitted first, and the remainder STRUCTURAL BIOLOGY of the structure was built directly into the den- sity. The final model consists of 2834 residues out of the 3469 total, with most of the missing Structure and flexibility of the residues predicted to be disordered. Although side chains were not visible at this resolution, the endosomal Vps34 complex reveals the sequence register was inferred from previously determined structures for most of Vps34, the basis of its function on membranes WD40 domain of Vps15, and the C-terminal do- main (CTD) of Vps30. An approximate sequence register was assigned for the remainder of the Ksenia Rostislavleva,1* Nicolas Soler,1* Yohei Ohashi,1 Lufei Zhang,1 Els Pardon,2,3 structure. The real-space correlation of the mod- John E. Burke,1† Glenn R. Masson,1 Chris Johnson,1 Jan Steyaert,2,3 el with the density suggests that the fit is rea- Nicholas T. Ktistakis,4 Roger L. Williams1‡ sonable for most of the structure. The poorest fit to the density is in the Vps38 N-terminal C2 Phosphatidylinositol 3-kinase Vps34 complexes regulate intracellular membrane domain (fig. S2). trafficking in endocytic sorting, cytokinesis, and autophagy. We present the 4.4 angstrom Overall architecture of complex II 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 The complex has a Y shape, with two long arms Y-shaped complex, centered on the Vps34 C2 domain. Vps34 and Vps15 intertwine in one and a short hook-like base (Fig. 1, D and E). The arm, where the Vps15 kinase domain engages the Vps34 activation loop to regulate its base is built entirely of the Vps30 and Vps38 N- activity. Vps30 and Vps38 form the other arm that brackets the Vps15/Vps34 heterodimer, terminal domains (NTDs) and coiled-coil 1 (CC1) suggesting a path for complex assembly. We used hydrogen-deuterium exchange mass domains. One of the arms (15 nm in length) spectrometry (HDX-MS) to reveal conformational changes accompanying membrane consists of Vps15 and Vps34 (Fig. 1E), whereas binding and identify a Vps30 loop that is critical for the ability of complex II to the other arm (18 nm) includes domains from all phosphorylate giant liposomes on which complex I is inactive. four subunits arranged along the Vps30 and Vps38 coiled-coil 2 (CC2). Vps30 and Vps38 show similar architectures except for their NTDs, he class III phosphatidylinositol 3-kinase signaling (7, 8), recycling (9), and lysosomal whereVps38hasaC2domain,whereasVps30 (PI3K) known as Vps34 (vacuolar protein tubulation (10). Independently from complex I is mostly unstructured (fig. S3, A and B). At the sorting 34, encoded by PIK3C3)ispresent and II, Beclin 1 and UVRAG also play separate C terminus, Vps30 has a BARA domain that T in all eukaryotes. It phosphorylates phos- roles in endosome function and neuron viability binds side-by-side to the CTD of Vps38; we named phatidylinositol (PI) to yield phosphatidy- (11). Elevated expression of PIK3C3, PIK3R4,and this domain BARA2. The two arms of complex linositol 3-phosphate (PI3P) (1), which is important BECN1 is associated with more aggressive forms II correspond to the V shapeseeninthelow- for all vesicle-mediated sorting pathways to lyso- of chronic lymphocytic leukemia (12), consistent resolution electron microscopy (EM) structure of somes, including processes such as phagocytosis, with autophagy being involved in tumor main- complex I (19). It is thus likely that most of the autophagy, multivesicular body formation, and tenance.