Chapter 5 - Approaches to the Study of Atg8-Mediated Membrane Dynamics In Vitro
Introduction
Autophagy is an evolutionarily conserved process that enables the sequestration of cytosol and cellular organelles within a double-membrane vesicle or autophagosome and facilitates its delivery to the lytic compartment (Klionsky, 2005, Mizushima et al., 2002, Nakatogawa et al., 2009). This action is exacerbated in response to intracellular stressors including nutrient deprivation and has been implicated in a variety of cell functions including organelle clearance, cell death, and tumor suppression.
How the autophagosome forms is only poorly understood. Much of the basic machinery was first characterized through yeast genetic approaches (Harding et al., 1995, Thumm et al., 1994, Tsukada and Ohsumi, 1993) and there are more than 30 autophagy-related (ATG) genes identified in S. cerevisiae. Biochemical approaches have segregated these gene products into different functional groups, but how these proteins act collaboratively to drive the membrane dynamics of autophagosome biogenesis has not been established (Xie et al., 2008b).
Of the ATG proteins identified, Atg8 is one of two ubiquitin-like proteins implicated in the formation of the autophagosome ((Geng and Klionsky, 2008) and Fig. 1). In yeast, Atg8 is synthesized with an arginine residue at its C terminus, which is subsequently removed by Atg4 to expose a glycine residue (Atg8G116) (Kirisako et al., 2000). In a ubiquitin-like conjugation reaction, E1-like and E2-like enzymes Atg7 and Atg3 couple this processed form of Atg8 to the lipid phosphatidylethanolamine (PE) (Ichimura et al., 2000) and the resulting proteo-lipid conjugate, Atg8-PE, is tightly associated with the autophagosomal membrane. Surprisingly, Atg8 is the only known stable, membrane-bound protein associated with the autophagosome, providing a reliable marker for the study of autophagy (Kirisako et al., 1999). Yeast lacking Atg8 form abnormally small autophagosomes (Abeliovich et al., 2000, Kirisako et al., 2000) as do starving yeast expressing attenuated amounts of Atg8 (Xie et al., 2008a), suggesting that a major function of this protein is to regulate membrane expansion. Mammalian cells depleted for a subset of Atg8 homologues by RNAi produce autophagosomes of different sizes (depending upon the subset present) (Weidberg et al., 2010), also consistent with a role in expansion. In addition, the loss of a subset of Atg8 also results in more omegasome-like structures, thought to be “open” autophagosomes (Weidberg et al., 2010). The same phenotype is observed if lipidation is blocked for all mammalian homologues (Fujita et al., 2008). These results indicate that the Atg8 family may also control a late step in autophagosome maturation that may include closure/fusion of the mature autophagosome. Using in vitro reconstitution of Atg8 lipidation, Ohsumi and colleagues recently established that Atg8 can tether liposomes, bringing the membranes into close apposition (Nakatogawa et al., 2007). They also concluded that Atg8 is itself a membrane fusogen, potentially establishing a new protein paradigm for membrane fusion machineries and providing the first mechanistic explanation for the various roles of the Atg8 family in cell biology (Nakatogawa et al., 2007). Our recent studies suggest that these in vitro functions of Atg8 are strongly dependent upon the design of the experiment (Nair et al., 2011), and additional studies may be required to establish whether Atg8 plays a direct fundamental role in membrane dynamics. Thus herein we describe these in vitro assays of Atg8 lipidation and function and discuss the strengths and limitation of each assay when exploring the potential direct role of the Atg8 family in autophagosome membrane dynamics.
Section snippets
Rationale
Our current understanding of the membranes (the Pre-Autophagosomal Structure (PAS) also termed phagophore or isolation membrane) to which Atg8 naturally couples is limited. Already in the 1960 s, electron microscopy revealed cup-like intermediate membrane structures in the autophagosome biogenesis events occurring in the liver of starved rats. A variety of experiments designed to block autophagosome maturation have yielded similar structures, sometimes termed omegasomes. Several models suggest
Rationale
Only three proteins are absolutely required for the lipidation reaction; co-expression in E. coli of a processed form of Atg8, Atg8G116 (Atg8 with a COOH-terminal deletion to expose the reacting glycine at position 116 – simply referred to as Atg8 in the rest of this manuscript) along with the enzymes Atg7 and Atg3 will result in lipidated Atg8 (Ichimura et al., 2004). To reconstitute completely in vitro, each protein can be individually expressed in and purified from bacteria (Ichimura et al.,
Rationale
Although Atg8 shares no sequence homology with ubiquitin, its lipidation reaction is perfectly analogous to the well-described coupling of ubiquitin to its substrate protein. The COOH-terminal glycine of ubiquitin reacts in an ATP-dependent manner with the reactive cysteine of its E1 enzyme to form a covalent sulfhydryl bond. The energy of this bond is used to transfer ubiquitin to the reactive cysteine of its E2 enzyme. The E2 enzyme brings ubiquitin and its substrate into close apposition and
Rationale
Although the enzymatic addition of PE to Atg8 is the most physiologically relevant, Atg8-driven membrane activities may also be revealed by artificial lipidation of Atg8 (Nair et al., 2011), or of its mammalian homologues (Ma et al., 2010, Weidberg et al., 2011). Such an approach may even present certain advantages when considering functions like membrane fusion (below) where either high PE concentrations or high enzyme concentrations can confound interpretation. This alternative
Rationale
Atg8 and its mammalian homologues are able to homo-multimerize in vitro (Nair et al., 2011, Nakatogawa et al., 2007, Nymann-Andersen et al., 2002, Pacheco et al., 2010, Weidberg et al., 2011). By gel electrophoresis, it is apparent that multimers of Atg8 subunits form and further, mutants that abrogate this multimerization also appear to abrogate autophagy (Nakatogawa et al., 2007). Multimers that form in the plane of the membrane via interactions that we would describe as “cis” have not yet
Conclusion
What then is the role of the Atg8 family in autophagosome maturation? Atg8 is properly situated spatially and temporally to impact the dramatic membrane rearrangements occurring in the later stages of autophagosome maturation. Precisely how Atg8 affects these maturation events remains contentious, but a variety of labs using complementary approaches are converging on some common themes including a clear capacity to tether membranes. This tethering suggests a trans-interaction state for the Atg8
Acknowledgments
Funding for this work was provided by a Kingsley Medical Fellowship (TJM), Yale Summer Fellowships including the STARS program, and the George J. Schulz Summer Fellowship in the Physical Sciences (AJ) and the REPU program – Research Experience for Peruvian Undergrads (OJZ). Cryo-electron microscopy was performed at the New York Structural Biology Center. We would also like to thank Shanta Nag and Dr. Sangeeta Nath for help in experiments.
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The coordination of membrane fission and fusion at the end of autophagosome maturation
2017, Current Opinion in Cell BiologyCitation Excerpt :In vitro reconstitution studies on lipid-anchored Atg8 proteins have suggested that they are membrane-active. Each can tether liposomes together into clusters [18–23,24•], and in some cases, support the mixing of lipids as either hemifusion or full fusion of lipid bilayers. Importantly the sequences that support these in vitro activities are also essential in cells.
Sensing Membrane Curvature in Macroautophagy
2017, Journal of Molecular BiologyCitation Excerpt :In a landmark paper on reconstitution, Atg8-PE was demonstrated to tether liposomes and even drive hemi-fusion [76]. Subsequent papers established similar activities for LC3, GATE-16, and GABARAP-L1 [77–80]. Fusion, however, appears to be limited to membranes of very high and non-physiological, cone-shaped lipid compositions as neither LC3-PE nor Atg8-PE is apparently fusion-active on other mixtures [69,73,78].