Kohichi Matsunaga

Two Beclin 1-binding proteins, Atg14L and Rubicon, reciprocally regulate autophagy at different stages

Beclin 1, a protein essential for autophagy, binds to hVps34/Class III phosphatidylinositol-3-kinase and UVRAG. Here, we have identified two Beclin 1 associated proteins, Atg14L and Rubicon. Atg14L and UVRAG bind to Beclin 1 in a mutually exclusive manner, whereas Rubicon binds only to a subpopulation of UVRAG complexes; thus, three different Beclin 1 complexes exist. GFP–Atg14L localized to the isolation membrane and autophagosome, as well as to the ER and unknown puncta. Knockout of Atg14L in mouse ES cells caused a defect in autophagosome formation. GFP–Rubicon was localized at the endosome/lysosome. Knockdown of Rubicon caused enhancement of autophagy, especially at the maturation step, as well as enhancement of endocytic trafficking. These data suggest that the Beclin 1–hVps34 complex functions in two different steps of autophagy by altering the subunit composition.

Atg9a controls dsDNA-driven dynamic translocation of STING and the innate immune response

Microbial nucleic acids are critical for the induction of innate immune responses, a host defense mechanism against infection by microbes. Recent studies have indicated that double-stranded DNA (dsDNA) induces potent innate immune responses via the induction of type I IFN (IFN) and IFN-inducible genes. However, the regulatory mechanisms underlying dsDNA-triggered signaling are not fully understood. Here we show that the translocation and assembly of the essential signal transducers, stimulator of IFN genes (STING) and TANK-binding kinase 1 (TBK1), are required for dsDNA-triggered innate immune responses. After sensing dsDNA, STING moves from the endoplasmic reticulum (ER) to the Golgi apparatus and finally reaches the cytoplasmic punctate structures to assemble with TBK1. The addition of an ER-retention signal to the C terminus of STING dampens its ability to induce antiviral responses. We also show that STING co-localizes with the autophagy proteins, microtubule-associated protein 1 light chain 3 (LC3) and autophagy-related gene 9a (Atg9a), after dsDNA stimulation. The loss of Atg9a, but not that of another autophagy-related gene (Atg7), greatly enhances the assembly of STING and TBK1 by dsDNA, leading to aberrant activation of the innate immune response. Hence Atg9a functions as a regulator of innate immunity following dsDNA stimulation as well as an essential autophagy protein. These results demonstrate that dynamic membrane traffic mediates the sequential translocation and assembly of STING, both of which are essential processes required for maximal activation of the innate immune response triggered by dsDNA.

Autophagy requires endoplasmic reticulum targeting of the PI3-kinase complex via Atg14L.

Generation of PI3P in the normally PI3P-deficient ER membrane makes the organelle a platform for autophagosome formation.

Modulation of local PtdIns3P levels by the PI phosphatase MTMR3 regulates constitutive autophagy

Autophagy is a catabolic process that delivers cytoplasmic material to the lysosome for degradation. The mechanisms regulating autophagosome formation and size remain unclear. Here, we show that autophagosome formation was triggered by the overexpression of a dominant‐negative inactive mutant of Myotubularin‐related phosphatase 3 (MTMR3). Mutant MTMR3 partially localized to autophagosomes, and PtdIns3P and two autophagy‐related PtdIns3P‐binding proteins, GFP‐DFCP1 and GFP‐WIPI‐1α (WIPI49/Atg18), accumulated at sites of autophagosome formation. Knock‐down of MTMR3 increased autophagosome formation, and overexpression of wild‐type MTMR3 led to significantly smaller nascent autophagosomes and a net reduction in autophagic activity. These results indicate that autophagy initiation depends on the balance between PI 3‐kinase and PI 3‐phosphatase activity. Local levels of PtdIns3P at the site of autophagosome formation determine autophagy initiation and the size of the autophagosome membrane structure.

Rubicon and PLEKHM1 negatively regulate the endocytic/autophagic pathway via a novel Rab7-binding domain.

Rubicon, a subunit of the Beclin 1-PI3-kinase complex and its homologue, PLEKHM1, negatively regulate endocytic pathway through the interaction with Rab7. Synchronous association with the Beclin 1–PI3-kinase complex and Rab7 is necessary for the function of Rubicon, but not PLEKHM1.

Regulation of membrane biogenesis in autophagy via PI3P dynamics

In autophagy, cytoplasmic substrates are targeted for degradation in the lysosome via membrane structures called autophagosomes. The formation of the autophagosome is the primary regulatory point for autophagy activity, and PI3P plays a central role in this process. In this review, we will discuss the role of PI3P in autophagosome formation from three different perspectives: PI3-kinase, PI3-binding proteins, and PI3-phosphatase. Recent developments in this field suggest that the local PI3P concentration is dynamically regulated during autophagy, and that this molecule is critical to the proper control of autophagy.

Binding Rubicon to cross the Rubicon.

Beclin 1 is an antitumor protein, required for mammalian autophagy, but its precise molecular function is poorly understood. Mass spectrometry analysis reveals that two novel proteins, Atg14L and Rubicon, associate with Beclin 1, together with a known Beclin 1-binding protein, UVRAG. The interactions of Atg14L and UVRAG with the Beclin 1-Vps34 (class III PI3-kinase)-Vps15 core complex are mutually exclusive; Rubicon associates with a subpopulation of UVRAG-containing complexes. The Atg14L complex, which positively regulates autophagy at an early step, localizes to the phagophore/isolation membrane, autophagosome and endoplasmic reticulum. In contrast, the Rubicon-UVRAG complex localizes to the late endosome/lysosome and negatively regulates both autophagy at a later step and the endocytic pathway. Thus, the Beclin 1-Vps34-Vps15 complex functions in autophagy and the endocytic pathway, but its function in a given context depends on the identity of its interacting subunits.

PI3K regulates endocytosis after insulin secretion by mediating signaling crosstalk between Arf6 and Rab27a

ABSTRACT In secretory cells, endocytosis is coupled to exocytosis to enable proper secretion. Although endocytosis is crucial to maintain cellular homeostasis before and after secretion, knowledge about secretagogue-induced endocytosis in secretory cells is still limited. Here, we searched for proteins that interacted with the Rab27a GTPase-activating protein (GAP) EPI64 (also known as TBC1D10A) and identified the Arf6 guanine-nucleotide-exchange factor (GEF) ARNO (also known as CYTH2) in pancreatic β-cells. We found that the insulin secretagogue glucose promotes phosphatidylinositol (3,4,5)-trisphosphate (PIP3) generation through phosphoinositide 3-kinase (PI3K), thereby recruiting ARNO to the intracellular side of the plasma membrane. Peripheral ARNO promotes clathrin assembly through its GEF activity for Arf6 and regulates the early stage of endocytosis. We also found that peripheral ARNO recruits EPI64 to the same area and that the interaction requires glucose-induced endocytosis in pancreatic β-cells. Given that GTP- and GDP-bound Rab27a regulate exocytosis and the late stage of endocytosis, our results indicate that the glucose-induced activation of PI3K plays a pivotal role in exocytosis–endocytosis coupling, and that ARNO and EPI64 regulate endocytosis at distinct stages. Summary: The insulin secretagogue glucose activates PI3K and induces endocytosis through promoting signaling crosstalk between Arf6 and Rab27a pathways.

Atg14L recruits PtdIns 3-kinase to the ER for autophagosome formation

Divergent phosphoinositides are generated to characterize specific organelles and recruit specific effector proteins to these sites. For example, phosphatidylinositol-3-phosphate (PtdIns(3)P) is a typical endosome marker and recruits many types of PtdIns(3)P binding proteins such as EEA1, Hrs, and sorting nexins, which are critical in endosomal functions. Likewise, the plasma membrane contains PtdIns(4,5)P2, whereas the Golgi complex has PtdIns(4)P. In this sense, the endoplasmic reticulum is known to be essentially free of phosphoinositide. In other words, this situation provides the ER with the opportunity to recruit whatever proteins are in demand. Recently, we have uncovered how PtdIns(3)P is generated on the ER for the autophagic process.

Rab2a and Rab27a cooperatively regulate the transition from granule maturation to exocytosis through the dual effector Noc2

ABSTRACT Exocytosis of secretory granules entails budding from the trans-Golgi network, sorting and maturation of cargo proteins, and trafficking and fusion to the plasma membrane. Rab27a regulates the late steps in this process, such as granule recruitment to the fusion site, whereas Rab2a functions in the early steps, such as granule biogenesis and maturation. Here, we demonstrate that these two small GTPases simultaneously bind to Noc2 (also known as RPH3AL) in a GTP-dependent manner, although Rab2a binds only after Rab27a has bound. In pancreatic β-cells, the ternary Rab2a–Noc2–Rab27a complex specifically localizes on perinuclear immature granules, whereas the binary Noc2–Rab27a complex localizes on peripheral mature granules. In contrast to the wild type, Noc2 mutants defective in binding to Rab2a or Rab27a fail to promote glucose-stimulated insulin secretion. Although knockdown of any component of the ternary complex markedly inhibits insulin secretion, only knockdown of Rab2a or Noc2, and not that of Rab27a, impairs cargo processing from proinsulin to insulin. These results suggest that the dual effector, Noc2, regulates the transition from Rab2a-mediated granule biogenesis to Rab27a-mediated granule exocytosis. Summary: Although regulated exocytosis comprises several sequential steps, the mechanisms coordinating each step are poorly understood. These findings suggest that Noc2 regulates the transition between Rab2a- and Rab27a-mediated exocytosis.