VPS34 complex

4. VPS34 complex

VPS34 (vacuolar protein sorting 34) gene was first discovered in a screening performed in Saccharomyces cerevisiae and was demonstrated to be essential for intracellular sorting of various vacuolar proteins (Herman and Emr, 1990). However, its

function as a lipid kinase was not elucidated until the significant homology between VPS34 and mammalian Class IA PI3K, p110α was uncovered (Schu et al., 1993).

VPS34 is classified as Class III PI3K, which produces PI3P from phosphatidylinositol (PI), and is highly conserved from yeast to mammalian cells. Its critical role in regulating endocytic trafficking and autophagy has been demonstrated in mouse model, in which VPS34 acts indispensably in sensory neuron, heart and liver, and T cells (Jaber et al., 2012; Parekh et al., 2013; Zhou et al., 2010).

4.1 Complex Composition and Cellular Function

VPS34 forms diverse multiple-subunits complexes in which VPS34-VPS15-Beclin1 composes the “core complex.” It is the additional proteins that combine with the core complex determine the cellular function of each VPS34 complex. UVRAG and ATG14L, homologs of VPS38 and ATG14 in mammalian cells, are two additional proteins that exist in distinct VPS34 complexes and constitute PI3K complex I and II, respectively (Matsunaga et al., 2009). Although studies performed in yeast reported that PI3K complex I is mainly involved in autophagy while complex II plays a role in vacuolar protein sorting pathway, both complexes are implicated in autophagy in mammalian cells (Kihara et al., 2001; Sun et al., 2010). The finding that depletion or ectopic expression of key members, such as VPS15 or VPS34, affects the protein level of other subunits suggests the interdependency of these subunits to maintain complex stability (Liu et al., 2011; Platta et al., 2012; Thoresen et al., 2010; Yan et al., 2009).

Currently, structure of yeast PI3K complex II was solved, which shed a light on the organization and differential activity of the two distinct complexes. The crystal structure

of PI3K complex II in yeast appears in a Y-shaped organization, with VPS15/VPS34 forming one arm in an antiparallel manner and the parallel VPS30/VPS38 heterodimer consisting of the other arm. In addition, VPS34 C2 domain stands as a central hub in the complex to engage all subunits. Though the structure of PI3K complex I remained unsolved, it is suggested that structure of these two PI3K complexes are very similar (Rostislavleva et al., 2015). The details of VPS34 core complex components, together with several peripheral binding proteins, will be discussed below:

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VPS15

VPS15, a putative Ser/Thr protein kinase also known as p150, is often regarded as the regulatory subunit of Class III PI3K (Herman et al., 1991). Studies have revealed that VPS15 is required for both PI3P production activity and interaction with additional proteins of VPS34 complex (Stack et al., 1993; Yan et al., 2009). Since the kinase activity of VPS15 has never been proofed, it is possible that VPS15 regulates VPS34 by interacting and modulating the active conformation of its kinase domain or stabilizing the protein but not phosphorylation (Backer, 2008; Rostislavleva et al., 2015). Besides, VPS15 may assist VPS34 targeting to a specific localization of cellular membrane via two mechanisms. First, an N-terminal myristoylation allows VPS15 to anchor on a membrane structure. Second, it possesses a WD40 domain that can interact with various proteins such as activated Rab5 on early endosomes, Rab7 on late endosomes, or even bridge between VPS15/34 and VPS30/VPS38 pairs in yeast (Christoforidis et al., 1999;

Rostislavleva et al., 2015; Stein et al., 2003).

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Beclin1

Beclin1, homolog of ATG6/VPS30 in mammalian cells, is another component of VPS34 core complex and contributes to the interaction with additional set of proteins.

In PI3K complex I and II, the central CCD domain of Beclin1 serves as a docking site for ATG14L or UVRAG (Liang et al., 2006; Sun et al., 2008). Furthermore, some other proteins may regulate autophagy by loosely or transiently interacting with Beclin1.

Among these peripheral proteins, the most famous example is a negative regulator of autophagy - anti-apoptotic Bcl2 family proteins. Through binging to the BH3 domain of Beclin1, Bcl2 proteins inhibit autophagy by interfering the interaction between Beclin1 and VPS34 (Pattingre et al., 2005). Ambra1, on the other hand, competes Bcl2 for binding Beclin1 (Strappazzon et al., 2011). Additionally, Ambra1 promotes Beclin1 ubiquitination, which increases Beclin1 interaction with VPS34 and VPS34 catalytic activity (Fimia et al., 2007).

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UVRAG

The UVRAG-containing VPS34 complex, mainly localized to endocytic compartments (Itakura et al., 2008), is the relatively dominant population and is known to participate in endocytic and autophagic pathways (Funderburk et al., 2010).

Endosomal fusion and autophagosome maturation are accomplished by two different populations of UVRAG that collaborate on regulating Rubicon, which is a negative regulator of autophagy and endocytic pathway and also a binding partner of UVRAG-VPS34 containing complex (Sun et al., 2010). While a population of UVRAG interacts with VPS34 complex, the other associates with Class C VPS complex, which is required for vesicle tethering and fusion (Itakura et al., 2008; Liang et al., 2008) . UVRAG-C-VPS complex on endosomes facilitates endosomal fusion and activates Rab7 by acting

as its GEF. GTP-loading enables Rab7 to compete for Rubicon binding with containing VPS34 complex, which finally abolish Rubicon’s sequestration of UVRAG-containing VPS34 (Sun et al., 2010).

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ATG14L

Although being regarded as a perquisite component that only exist in autophagy-specific VPS34 complex, the mammalian ortholog of ATG14 has not been identified until 2008 (Sun et al., 2008). Despite the functions of ATG14L have not been well characterized, it was shown that ATG14L resembles its yeast homolog by directing VPS34 complex to the site where autophagosomes originally form (Obara and Ohsumi, 2011). To recruit VPS34 complex onto the omegasome, both the N-terminal conserved cysteine repeats and C-terminal Barkor/Atg14(L) autophagosome targeting sequence (BATS) domain are essential (Fan et al., 2011; Matsunaga et al., 2010). The cysteine repeats determine the localization of VPS34 complex whereas BATS domain senses membrane curvature and preferentially incorporates to the PI3P-enriched membrane through the hydrophobic surface of an intrinsic amphiphilic helix, leading to enhanced binding and stabilization of membrane curvature (Fan et al., 2011; Matsunaga et al., 2010). Interestingly, several proteins, such as RACK1, Dapper1, PAQR3 and SLC35D3, were reported to promote autophagy by enhancing ATG14L-associated PI3K complex formation under various conditions (Ma et al., 2014; Wei et al., 2016; Xu et al., 2016;

Zhao et al., 2015), while Nrbf2 modulates complex formation conversely (Zhong et al., 2014).

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4.2 Regulation of VPS34

Compared to other components such as Beclin1 and ATG14L in the complex, regulatory process on VPS34 by post-translational modification is relatively rare. To date, studies have revealed that VPS34 could be regulated by phosphorylation, ubiquitination, and sumoylation.

Through phosphorylation, kinase activity and complex composition of VPS34 could be fine-tuned under various conditions. The key energy sensor AMPK was demonstrated to regulate distinct VPS34 complexes upon glucose starvation. Among multiple VPS34 complexes, non-autophagy VPS34 complex is suppressed by AMPK through phosphorylating VPS34 at Thr163/Ser165 to protect cells from starvation (Kim et al., 2013). During mitosis, the mitotic kinase CDK1 phosphorylates VPS34 at Thr159, which reduces VPS34 lipid kinase activity through disrupting its interaction with Beclin1 (Furuya et al., 2010). The other member of CDK family - CDK5, a neuronal CDK that functions during neuronal development as well as neurotransmitter signaling in mature nervous system, phosphorylates VPS34 at Thr668, leading to the inhibition of VPS34 lipid kinase activity (Furuya et al., 2010). Moreover, the interplay between phosphorylation and ubiquitination also plays a role in regulating VPS34.

When cells encounter DNA damage, level of FBXL20, an adaptor of Cul1-Skp1 E3 ligase complex, is induced in a p53-dependent manner and the following mitotic arrest leads to the accumulation of cyclin B1 that activates CDK1. Phosphorylation of VPS34 by CDK1 facilitates its recognition by Cul1-FBXL20, which targets VPS34 for ubiquitination and proteasomal degradation, thereby downregulates autophagy (Xiao et al., 2015).

Ubiquitination, as mentioned, is a complicated modification for its competence to form various chain types. However, VPS34 has only been reported to be modified

with the degradative ubiquitin chains. In addition to Cul1-FBXL20, previous work of our lab also discovered that another E3 ligase, Cul3-KLHL20, displays an enhanced binding with VPS34 upon prolonged starvation and mediates its ubiquitination and degradation (Liu et al., 2016). Deubiquitination and stabilization of VPS34 has also been addressed in the study referring a small molecular autophagy inhibitor Spautin-1, which interferes with the deubiquitinating function of USP10/13 and causes destabilization of VPS34 and other components in the complex (see section 3.2) (Liu et al., 2011).

Sumoylation is also involved in VPS34 regulation. Under stress-induced condition, increased intracellular acetylated HSP70 binds to VPS34-Beclin1 complex and recruits SUMO E3 ligase - KAP1. KAP1 mediates Lys840 sumoylation on VPS34, which strengthens the interaction between VPS34 and Beclin1 (Yang et al., 2013).

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