In the present study, we have demonstrated that Bif-1 regulates the fission of Golgi membranes and the trafficking of Atg9 from the Golgi complex to autophagosomes during starvation. In response to nutrient starvation, Atg9-positive Golgi membranes were dispersed throughout the cytoplasm where they colocalized not only with Bif-1, but also with LC3 and Atg16L. Moreover, loss of Bif-1 resulted in the suppression of Golgi fission, Atg9 redistribution and autophagosome formation during starvation. These observations suggest that Atg9-positive membranes derived from the Golgi complex may contribute to the formation and/or expansion of autophagosome precursors. Indeed, our previous time-lapse studies show that an Atg9-Bif-1 positive punctum fuses with another Atg9-Bif-1 small membrane during the formation of a ring-shaped structure.26
It has been shown that Atg9 self-interaction is required for expansion of autophagosome precursors in yeast35
and that loss of Atg9 suppresses the lipidation of LC3 I and/or LC3 foci formation.15,20,21
Moreover, recent studies in yeast have shown that proteins involved in the secretary pathway at the Golgi complex play a key role in autophagosome formation.11,12
In addition, it has been reported that autophagosomes are formed through the fusion of isolation membranes with vesicles derived from the Golgi complex and late endosomes independent of Atg5/Atg7 in mammalian cells.10
These studies, taken together with our results, strongly support the notion that Golgi-derived membranes contribute to the formation of autophagosomes. However, the phenomenon of Golgi fragmentation after starvation has not been reported in previous studies.13,20
This discrepancy could be due to the difference in cell lines and/or starvation media used to induce autophagy. Interestingly, we also found that a portion of Atg9 colocalizes with a marker of early endosomes, Rab5. As plasma membrane that is internalized through clathrin-mediated endocytosis has been shown to regulate the early stages of autophagosome formation,14
we do not exclude the possibility that Atg9-positive structures used for the formation of pre-autophagosomal structures are derived from early endosomes.
The precise molecular machinery through which Bif-1 regulates the fission of Atg9-positive Golgi membranes during the induction of autophagy remains to be determined. However, our results demonstrate that loss of either the H0 or H1I region of the Bif-1 N-BAR domain suppresses Bif-1-mediated Atg9 puncta formation. As these regions are involved in membrane binding and tubulation,32,33
we propose a model in which Bif-1 regulates the redistribution of Atg9 from the Golgi complex to peripheral sites by deforming Golgi membranes into narrow tubules to facilitate membrane fission. Although the fission of Atg9-positive Golgi membranes occurs in a Bif-1-dependent manner during starvation, the morphology of the Golgi complex under normal culture conditions and the disassembly of the Golgi complex during mitosis are not affected by loss of Bif-1. Moreover, while loss of COPI causes fragmentation of the Golgi complex, it does not promote the formation of autophagosomes.36
Thus, the Golgi fission machinery, which generates Atg9-containing membranes for autophagosome formation, is uniquely controlled by Bif-1 during nutrient starvation.
Activation of PI3KC3 is essential for the trafficking of Atg9 and the formation of autophagosomes in both yeast and mammalian systems.20,37
Consistently, our results indicate that inhibition of the Beclin 1-PI3KC3 complex prevents Atg9 foci formation after nutrient starvation. Although the precise mechanism by which the activation of PI3KC3 regulates Atg9-positive foci formation is not fully understood, PI3P produced by this lipid kinase plays an important role in the recruitment of proteins required for Atg9 trafficking.6,38
In fact, it has been reported that production of PI3P is required for the autophagosomal localization of mAtg18/WIPI-1,39
the mammalian orthologue of yeast Atg18, which interacts with Atg9 and is required for Atg9 trafficking to the pre-autophagosomal structure.37
As starvation promotes Bif-1 translocation to Atg9-enriched fractions, it will be interesting to determine whether production of PI3P facilitates Bif-1 localization to fission sites for the generation of Atg9 vesicles. Our results also show that knockdown of UVRAG suppresses starvation-induced redistribution of Atg9 during starvation. Thus, UVRAG may be involved in the regulation of Atg9 trafficking during the induction of autophagy. However, we did not observe any significant colocalization of UVRAG with Atg9 foci after starvation, suggesting that UVRAG dissociates from Atg9-positive punctate structures after the fission event occurs. Clearly, further studies are required in order to determine the role of the PI3KC3 complex II in Atg9 trafficking.
Despite significant inhibition of autophagosome formation by loss of Bif-1 in our cell culture systems, the phenotypes of Bif-1-deficient mice are quite different from the embryonic lethal phenotype observed in mice lacking Beclin 1.22,40,41
This suggests that other proteins, such as its family member Endophilin B2, may functionally compensate for the loss of Bif-1 during embryonic development. Indeed, our unpublished data indicate that Endophilin B2 also localizes to the Golgi complex and interacts with UVRAG. Further studies on the characterization of Bif-1 and Endophilin B2 double knockout mice will allow insight into how these proteins work together in the regulation of Golgi remodeling and autophagosome biogenesis during development and tissue homeostasis. Nevertheless, this study has shown for the first time that the N-BAR domain-containing protein, Bif-1 plays an essential role in the fission of the Golgi complex during nutrient starvation presumably to deliver Atg9-positive membranes for autophagosome formation.