A number of reports suggest that there is a connection between mitochondria and autophagosome formation. Several autophagy proteins (e.g., Beclin-1, the yeast protein Atg9p, a proteolytic fragment of Atg5, and Bif-1) have been previously localized to mitochondria (Reggiori et al., 2005
; Takahashi et al., 2007b
; Yousefi et al., 2006
). Additionally, several mitochondrial-localized proteins (e.g., smARF, Bif-1/EndophillinB) positively regulate autophagy (Lee et al., 2008
; Reef et al., 2006
; Takahashi et al., 2007a
). Knockdown of Bif-1 (a Bax-binding protein that also binds the Class III PI3Kinase complex) suppresses induction of autophagy during starvation, as does knockdown of Sirt1, a deacetylase that regulates mitochondrial biogenesis by deacetylating PGC1α. While reports of interplay between autophagy and mitochondria are often ascribed to mitophagy, our data suggest an alternative—that mitochondria participate in the formation of autophagosomes. Here, we show that autophagosome inception occurs along mitochondria. The early autophagosomal marker mApg5 transiently localizes to punctae on the mitochondria. LC3 subsequently replaces mApg5 at these sites where LC3-positive autophagosomes transiently associate with mitochondria. We show that a tail-anchored outer mitochondrial membrane protein labels the membranes of autophagosomes, and that its delivery to autophagosomal membranes is via the mitochondria membrane. We demonstrate this marker can be depleted from autophagosomes by photobleaching associated mitochondrial elements; therefore the membranes of these organelles are transiently shared. We further show that a mitochondrial lipid probe is conveyed from mitochondria to autophagosomes. Tomography and immuno EM further reveal autophagic structures associated with mitochondria that exclude mitochondrial matrix and inner membrane. Finally, we show that no autophagosome induction during starvation occurs in cells lacking MFN2, a mitochondrial protein involved in tethering of mitochondria to ER.
Formation of autophagosomes from the outer mitochondrial membrane requires delivery of the most upstream autophagic factors to the mitochondrial membrane. Consistent with this, Beclin-1 (a component of the Vps34/Beclin Class III PI3Kinase complex) has been reported on mitochondria. One plausible scenario by which multilamellar structures could derive from the outer membrane involves recruiting autophagy machinery to the mitochondria in order to impose membrane curvature on the outer mitochondrial membrane (see schematic, ). The Vps34/Beclin complex may mark an initiation site. Phosphorylation of the target phosphoinositide and Bif-1 BAR domain interactions with membrane could create and stabilize a microdomain that subsequently recruits the mApg5/Atg12/Atg16 complex. Oligomerization of this complex (reported in (Kuma et al., 2002
; Mizushima et al., 2003
) could then form a transient coat and expand the initiation site. LC3 conjugation to phosphotidylethanolamine (PE) at the site could stabilize local high concentrations of PE in the outer leaflet of the mitochondria outer membrane and support continued outgrowth of a structure. Notably, PE is one of a small set of lipids that imposes a negative radius of curvature, favoring formation of a cup-like structure that could capture cytosol within its volume (Thomas and Poznansky, 1989
; van Meer et al., 2008
). A multi-lamellar structure could then form if the distal edges fused. LC3 has been shown to catalyze fusion of homotypic membranes in an in vitro
system (Nakatogawa et al., 2007
). Such a scenario is consistent with the known activities of core autophagy machinery, and would allow for the establishment of asymmetric lipid composition from an existing membrane source to promote membrane curvature.
Given the ability of autophagy machinery to target a membrane, establish a stable microdomain, and promote membrane curvature, autophagosomes could potentially form from a variety of sources. Core autophagy machinery, which is almost entirely cytosolic, has been reported to target membranes of different origins. While ER stress-induced autophagosomes appear to utilize ER membrane (Bernales et al., 2006
), other reports have recently demonstrated that the autophagy protein LC3 can also be recruited to membranes derived from the plasma membrane (Sanjuan et al., 2007
). We therefore postulated that inducing autophagy in our line by another stress might produce autophagosomes with characteristics that are distinct from autophagosomes induced by starvation. A number of conditions have been reported to induce autophagy, including ER stress. When we treated NRK58B cells with the ER calcium pump inhibitor thapsigargin to perturb the ER folding environment (Brostrom and Brostrom, 2003, Tadini-Buoninsegni et al., 2008), CFP-LC3 positive structures were robustly generated, consistent with other reports or ER stress-induced autophagy (Ogata et al., 2006; Sakaki et al., 2008). Notably, thapsigargin induced structures were sensitive to 3-MA. However, they did not label with YFP-Mitocb5
TM (see Supplemental Fig. 3s
). Utilization of different membranes in autophagosome biogenesis thus may produce different classes of autophagosomes, resulting in autophagic structures with behaviors specific to their induction conditions (see Supplemental Fig 6s
). We favor the idea that the question of what membrane is utilized collapses to a question of how autophagosome assembly is initiated at diverse sites.
Why might starvation specifically utilize mitochondrial membrane? A little-explored aspect of autophagy is its potential role in fluxing lipids through otherwise disconnected cellular compartments. Our photochase data employing LC3 tagged with photoactivatable GFP indicates that a significant amount of membrane is moving from the autophagosomal origin to autolysosomes/lysosomes via fusion of outer autophagosomal membranes with lysosomal membranes. The lipid target of LC3—phosphotidylethanolamine—is an abundant cellular lipid that is transferred by autophagosomes. PE is synthesized principally at two sites—in the ER via the CDP-ethanolamine pathway and in the mitochondria via decarboxylation of phosphotidylserine (Vance, 2008
). ER synthesis of PE utilizes DAG and exogenous ethanolamine. By contrast, synthesis in the mitochondria utilizes phosphotidylserine transferred from the ER. Under starvation conditions the sources for exogenous ethanolamine and DAG (produced following growth factor engagement) are restricted. These substrates are required for PE synthesis in the ER. Autophagy may counter this by routing mitochondrial-derived PE to lysosomes, and subsequently via retrograde transport, back through the secretory pathway. Mitochondria contribution to autophagosomal membranes under starvation conditions may therefore contribute to lipid homeostasis in addition to established roles in nutrient recycling.
Given a significant flux of lipid, one prediction of utilizing mitochondrial lipids is a depletion of mitochondria. However, starvation-induced autophagy does not result in net loss of mitochondrial mass in our system. In fact, mitochondrial mass increases slightly with increased incubation time in DPBS. Other conditions reported to induce autophagy (i.e., Sirt1 overexpression) similarly show concurrent autophagosome proliferation and increased mitochondrial mass (Lee et al., 2008
). How might mitochondrial mass be maintained? Notably, a major source for mitochondrial phospholipids is the ER. Mitochondrial-associated membranes (MAM’s) act as bridges between the mitochondria and ER in both yeast and mammals, where phosphotidylserine and other phospholipids are transferred to mitochondria (Achleitner et al., 1999
; Bozidis et al., 2008
). An unexplored question is whether autophagy promotes transfer of lipid from the ER to mitochondria. We have evaluated whether ER/mitochondrial connections are important for autophagic induction. de Brito and Scorrano recently reported that loss of mitofusin2 disrupts connections between the ER network and mitochondria. We found that mitofusin2 knockout cells fail to produce autophagosomes when starved. Therefore, there appears to be a requirement for lipid contribution from the ER to mitochondria in order for starvation-induced autophagy to proceed.
Work by Axe et al reported that DFCP1, a unique PI3(P) binding protein in the ER, translocates to punctae under starvation that label sites where autophagosome formation occurs (Axe et al., 2008
). The authors observe mApg5 and LC3 autophagosomal markers surrounded by DFCP1, and present a model suggesting autophagosomes form from ER membrane at these sites. Our data suggest another possibility—that DFCP1 sites may be sites of connection between the ER and mitochondria that respond to starvation conditions. Conditions that induce the very rapid formation of autophagosomes may drive transfer of lipid from the ER to the mitochondria where these lipids are subsequently modified and utilized in autophagosome biogenesis without affecting mitochondrial mass.
Whether autophagosomes form de novo
or from pre-existing cytomembranes is a long-standing debate, and investigating how autophagosome assembly is initiated and proceeds remains difficult (Juhasz and Neufeld, 2006
). The findings presented here indicate that mitochondria participate directly in the formation of autophagosomes while retaining mitochondrial proteins by diffusion barriers. This utilization of the outer membrane of the mitochondria defines a new intracellular pathway from mitochondria to the autophagosomal/lysosomal system. Budding of vesicles from mitochondria that transit to peroxisomes has been reported (Neuspiel et al., 2008
). Such events, together with the mitochondrial pathway to autophagosomes reported here, may underlie the constitutive movement of mitochondrial-derived factors (e.g., heme (Rajagopal et al., 2008
)) to different cellular locations, and implicate mitochondrial involvement in as yet unappreciated aspects of cell biology.