In recent years, the degradative pathway of autophagy has attracted great interest because of its involvement in many physiological processes and its induction in numerous pathological situations; however, several important questions are still unanswered. One of the most intriguing concerns the origin of the lipid bilayer used to create the double-membrane vesicles, the hallmark of this transport route. Localization studies of most Atg proteins in different model organisms have furnished no clues because these factors are principally cytosolic and only transiently associate with autophagosomes and the PAS. Accordingly, we have focused on Atg9, the only identified integral membrane protein essential for autophagosome formation. 22
We have recently shown that in S. cerevisiae
this protein shuttles between the PAS and several cytoplasmic punctate sites. 7
In the present study we discovered that this punctate localization corresponds in part to mitochondria in both S. cerevisiae
and P. pastoris
revealing an undiscovered function for this organelle (; Figs. and ; ; and ). It should be noted however, that the PAS is not part of the mitochondrial network (). Connections between autophagy and mitochondria have been previously made only in the yeast Hansenula poly-morpha
undergoing pexophagy and in macrophages infected by the bacterium Legionella pneumophila
In both cases, the cargo being enwrapped within the double-membrane vesicle is surrounded by mitochondria prior to sequestration. Our data, however, cannot rule out the possibility that Atg9 is in a novel membranous structure closely attached to the outside of the mitochondria.
Atg9 was observed both on the mito-chondria and on the PAS supporting the model that this factor shuttles between these two locations (); 7
however, we cannot exclude the possibility that a population of Atg9 localizes to another structure or organelle for which we do not have adequate marker proteins. The function of Atg9 cycling is not clear, but one possibility is that it is required to supply the forming autophagosomes, at least in part, with membrane. This idea is supported by the fact that Atg9 was always associated with lipid bilayers and some of the Atg9 aggregates were cytosolic (; Figs. and ; and ). The ER is the principal lipid factory in the cell but mitochondria actively participate in the synthesis and modification of some lipids. The mitochondria-associated membranes (MAM) represent this close relationship between these two organelles in regard to lipid processing. MAM are regions of the ER that are in close juxtaposition to the mitochondrial outer membrane and act as a conduit for the transfer of lipids. 31
Thus, mitochondria are a potential source membrane for the autophagosome. Atg9 however, did not appear to localize to the MAM (). Even if the “lipid supply” model is correct, we cannot exclude that Atg9 carries out other functions. Under certain conditions, autophagy becomes the principal source of energy for the cell. Because the mitochondria provide the other primary supply of energy, one could imagine that Atg9 is used to coordinate the two sources.
Another aspect of Atg9 localization revealed by our work is that this protein was not homogeneously distributed along the entire mitochondrial surface but was concentrated to discrete areas that have a high mobility in between the organelle boundaries (; Fig. , and ). The observation that Atg9 forms clusters is consistent with our IEM analysis of this protein as well as a previous study (). 22
So far, there are no reports describing the presence of microdomains or specialized regions on the mitochondrial outer membrane. Our biochemical data indicate that one explanation for Atg9 clustering is its ability to self-associate (). However, our two-hybrid analysis in yeast and the coimmunoprecipitation experiment cannot exclude the possibility that one or more proteins that are present in these aggregates bridge Atg9 homo-association.
The majority of the Atg9 complexes were on the mitochondrial network (). After subcellular fractionation, however, only a relatively small amount of this protein was found bound to this organelle (). Thus cell lysis seems to provoke Atg9 dissociation from mitochondria. This loss of binding could be caused by the stimulation of the Atg9 sorting event. Alternatively, release of Atg9 could be due to the peripheral association of certain Atg9 aggregates. Importantly, the released material is associated with lipid () and further stripping of the mitochondria with high salt released additional membrane bound Atg9 (our unpublished data). All together, these findings support the idea that Atg9 leaves mitochondria in association with membranous structures, possibly vesicles or small cisternae. It is interesting to notice that the Atg9-containing clusters released from mitochondria or present in the cytosol (S13 supernatant fraction) have a very similar density as that of the PAS because they cofractionate on density gradients. 26
During the preparation of this manuscript, a report was published that demonstrates that the two human proteins with high homology to Atg9, HsAtg9L1 and HsAtg9L2, are its functional orthologues. 32
The yeast P. pastoris
is evolutionarily close to S. cerevisiae
and not surprisingly Atg9 localization in this organism was almost identical. In contrast, our preliminary results with mammalian cells indicate that HsAtg9L1 32
was not distributed on the mitochondrial network indicating that higher eukaryotes could supply autophagy with membranes by extracting lipids from a different reservoir (data not shown). This observation could also explain why HsAtg9 cannot substitute for the yeast Atg9 (data not shown). 32
In human adult tissues, HsATG9L1
is ubiquitously expressed whereas HsATG9L2
is highly expressed in placenta and pituitary gland. Importantly, the authors showed that these two factors are not distributed on mitochondria but to a perinuclear region in complete support of our preliminary observations. It is interesting to note, however, that HsAtg9L2 possesses a non-functional mitochondrial targeting sequence that is also present in its closest higher eukaryote homologues. 32
This characteristic raises the possibility that this is an ancient localization signal.
What is the role of Atg9-containing membranes during autophagosome biogenesis? In the current model, the elongation of a small template membrane, termed the isolation membrane or phagophore, leads to autophagosome formation in mammalian cells (). 22
The surface of this small cisterna is decorated with Atg16 and Atg5. 37
Conjugation of the ubiquitin-like Atg12 to Atg5 seems to be the induction step that triggers the elongation of this compartment. It is not known how the Atg12-Atg5 complex recruits additional lipid bilayers but the crescent autophagosome acquires more Atg12-Atg5 as well as a second ubiquitin-like molecule, Atg8/LC3, that is unconventionally linked to phosphatidylethanolamine. In yeast, the PAS very likely represents this growing cisterna. In the absence of Atg9, Atg5 is not targeted to the PAS and accordingly Atg8/LC3-containing membranes cannot be recruited to this location, thus double-membrane vesicles are not formed. 6
Atg8-containing membranes seem to have a different origin than the mitochondria, possibly the Golgi apparatus. 26
Figure 7 Putative model for Atg9 trafficking in yeast S. cerevisiae. Atg9-containing membranes are derived from mitochondria and become an isolation membrane or phagophore by acquiring Atg16 and Atg5. The covalent (more ...)
Our work has revealed Atg9 to be the first Atg component that localizes to a distinct subcellular compartment making it the first marker that allows monitoring at least part of the lipid bilayers used during autophagosome assembly. Future work on this factor will help to unveil the mode of membrane transport to and from the PAS. In addition, identification of proteins involved in the sorting of Atg9 from the mitochondria, or present in the Atg9 aggregates, will help to explain how autophagosomes are formed and potentially also reveal how their lipid bilayers are derived from the membrane origin, providing a model for the study of the same process in higher eukaryotes.