Research Article

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

+ See all authors and affiliations

Science  09 Oct 2015:
Vol. 350, Issue 6257, aac7365
DOI: 10.1126/science.aac7365

You are currently viewing the abstract.

View Full Text

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

Structured Abstract


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.


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.


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.


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.

View Full Text