|Home | About | Journals | Submit | Contact Us | Français|
Autophagosomes, the hallmark of autophagy, are double-membrane vesicles sequestering cytoplasmic components. They are generated at the phagophore assembly site (PAS), the phagophore being the precursor structure of these carriers. According to the current model, autophagosomes result from the elongation and reorganization of membranes at the PAS/phagophore driven by the concerted action of the autophagy-related (Atg) proteins. Once an autophagosome is completed, the Atg proteins that were associated with the expanding phagophore are released in the cytoplasm and reused for the biogenesis of new vesicles. One molecular event required for autophagosome formation is the generation of phosphatidylinositol 3-phosphate (PtdIns3P) at the PAS. Our data indicate that in addition to the synthesis of this lipid, the dephosphorylation of PtdIns3P is also crucial for autophagy progression. In the absence of Ymr1, a specific PtdIns3P phosphatase and the only yeast member of the myotubularin protein family, Atg proteins remain associated with complete autophagosomes, which are thus unable to fuse with the vacuole.
Our knowledge about the function and dynamics of PtdIns3P in autophagy is limited. Phosphorylation of phosphatidylinositol at position 3 of the inositol ring is an early event during autophagosome formation that occurs at the PAS by the action of the PtdIns 3-kinase Vps34, which is recruited to this structure as part of the protein complex PtdIns 3-kinase complex I. Reduction of PtdIns3P levels at the PAS prevents vesicle formation probably because Atg proteins directly or indirectly binding to this lipid fail to associate with this structure and remain cytosolic. Studies from the Deretic laboratory have revealed that PtdIns3P phosphatases modulate autophagy in a similar way. Members of the myotubularin protein family act as negative regulators of this pathway by inhibiting the recruitment of PtdIns3P-binding ATG proteins to autophagosomal membranes. As a consequence, regulation of PtdIns3P levels at the PAS by the action of lipid kinases and phosphatases could be crucial during autophagosome initiation by modulating the recruitment and activity of Atg proteins. PtdIns3P levels are probably also important at later stages of vesicle biogenesis, such as membrane elongation and autophagosome completion. Our data in particular show that autophagy is severely compromised in the absence of YMR1 or in cells expressing a phosphatase dead mutant of Ymr1. Under these conditions, autophagosomes are still formed but Atg proteins remain associated with them, impairing their fusion with the vacuole. These observations indicate that dephosphorylation of PtdIns3P by Ymr1 has an important role at the late stages of autophagosome biogenesis by mediating the release of Atg proteins from the surface of autophagosomes and possibly directly or indirectly triggering the recruitment and/or activation of the fusion machinery. This event thus appears to be a prerequirement for autophagosome fusion with vacuoles and it could act as a checkpoint to avoid the fusion of precursor structures/incomplete vesicles.
PtdIns3P levels on autophagosomal membranes are regulated in mammals by the action of more than one phosphoinositide phosphatase. Functional redundancy between these enzymes appears to exist also in yeast because autophagy is not completely abrogated in the absence of Ymr1; e.g., autophagosomes are still formed and fuse with the vacuole, but to a much lower extent than in wild-type cells. We have found that Inp53/Sjl3 also localizes to the PAS and therefore it is probable that Inp53 together with Ymr1 participates in specific aspects of PtdIns3P turnover. While the inp53 mutant strain does not display a defect in autophagy, this pathway is completely blocked in the double knockout ymr1 inp53, which could indicate that Inp53 is partially compensating for the absence of Ymr1. According to our unpublished data, however, this double mutant is unable to generate autophagosomes. In ymr1 inp53 cells the autophagosmal protein marker Atg8 is distributed to several cytoplasmic puncta. These structures are not autophagosomes, but rather pre-autophagosomal intermediates/precursors because they persist when ATG1 is additionally deleted. This observation suggests that PtdIns3P turnover could also be important during autophagosome initiation, a notion supported by the early recruitment of Ymr1 to the PAS and the fewer autophagosomes formed in ymr1 cells compared with the wild type. Alternatively, the ymr1 inp53 double knockout displays severe endosomal trafficking defects and this could indirectly impair some of the functions of the Golgi and/or the Atg9-containing compartments; both organelles play a key role in autophagosome biogenesis. Additionally, this mutant displays elevated levels of PtdIns3P compared with the wild type, which could lead to the misregulation of autophagy by affecting specific signaling cascades.
Interestingly, control of PtdIns3P levels by the action of phosphoinositide kinases and phosphatases during organelle biogenesis is not unprecedented. Recruitment to the endosomal membranes of various factors including the retromer, a complex involved in retrieval of specific proteins, and the ESCRT machinery, which mediates the formation of intralumenal vesicles, relies on the synthesis of PtdIns3P by the PtdIns 3-kinase complex II, which also contains Vps34. These components assist the maturation of endosomes into multivesicular bodies (MVBs). Similarly to autophagosome biogenesis, MVB completion and subsequent fusion with the vacuole/lysosome requires the turnover of PtdIns3P from their surface. Consumption of this lipid at the MVBs occurs through three different mechanisms: hydrolysis by phosphatases such as Ymr1, conversion into PtdIns(3,5)P2 by the Fab1/PIKFYVE kinase, and internalization of PtdIns3P into MVB vesicles, which topologically correspond to the inner membrane of autophagosomes. While Fab1 does not appear to play a role in autophagy, studies from the Ohsumi and Proikas-Cezanne laboratories have revealed that PtdIns3P is specifically enriched in the autophagosome internal membrane. Thus, it appears that the late endosome and autophagosome maturation processes are regulated not only through a similar mechanism but also through common factors.
Regulation of PtdInd3P levels on autophagosomal membranes thus seems to be a process of great complexity. At a temporal level, dynamics of this lipid could be important to regulate the function of the Atg machinery by controlling the recruitment and dissociation of specific proteins at different steps of the autophagosome formation process. Spatially, PtdIns3P dynamics could be differently regulated on the distinct areas/surfaces of the nascent autophagosome. This spatio-temporal modulation of the PtdIns3P levels could allow proteins to be selectively recruited at specific action sites, or to drive membrane shaping during autophagosome formation. The future investigation of these putative scenarios is necessary to completely understand the role(s) of PtdIns3P dynamics during autophagy (Fig. 1).
The authors thank Jana Sanchez Wandelmer for critically reading the manuscript. F.R. is supported by ECHO (700.59.003), ALW Open Program (821.02.017) and DFG-NWO cooperation (DN82) grants. E.C. is supported by an ALW Open Program grant (817.02.023).
Cebollero E, van der Vaart A, Zhao M, Rieter E, Klionsky DJ, Helms JB, et al. Phosphatidylinositol-3-phosphate clearance plays a key role in autophagosome completion Curr Biol 2012 22 1545 53 doi: 10.1016/j.cub.2012.06.029.
Previously published online: www.landesbioscience.com/journals/autophagy/article/22162