Chapter 5 - Approaches to the Study of Atg8-Mediated Membrane Dynamics In Vitro

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Abstract

Macro-autophagy is the intracellular stress-response pathway by which the cell packages portions of the cytosol for delivery into the lysosome. This “packaging” is carried out by the de novo formation of a new organelle called the autophagosome that grows and encapsulates cytosolic material for eventual lysosomal degradation. How autophagosomes form, including especially how the membrane expands and eventually closes upon itself is an area of intense study. One factor implicated in both membrane expansion and membrane fusion is the ubiquitin-like protein, Atg8. During autophagy, Atg8 becomes covalently bound to phosphatidylethanolamine (PE) on the pre-autophagosomal membrane and remains bound through the maturation process of the autophagosome. In this chapter, we discuss two approaches to the in vitro reconstitution of this lipidation reaction. We then describe methods to study Atg8-PE mediated membrane tethering and fusion, two functions implicated in Atg8's role in autophagosome maturation.

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.

References (63)

  • L.D. Mayer et al.

    Vesicles of variable sizes produced by a rapid extrusion procedure

    Biochim. Biophys. Acta

    (1986)
  • U. Nair et al.

    SNARE Proteins Are Required for Macroautophagy

    Cell

    (2011)
  • H. Nakatogawa et al.

    Atg8, a ubiquitin-like protein required for autophagosome formation, mediates membrane tethering and hemifusion

    Cell

    (2007)
  • R. Nebauer et al.

    Phosphatidylethanolamine, a limiting factor of autophagy in yeast strains bearing a defect in the carboxypeptidase Y pathway of vacuolar targeting

    J. Biol. Chem.

    (2007)
  • J. Nymann-Andersen et al.

    Biochemical identification of the binding domain in the GABA(A) receptor-associated protein (GABARAP) mediating dimer formation

    Neuropharmacology

    (2002)
  • K. Oh-oka et al.

    Physiological pH and acidic phospholipids contribute to substrate specificity in lipidation of Atg8

    J. Biol. Chem.

    (2008)
  • B.L. Scott et al.

    Liposome fusion assay to monitor intracellular membrane fusion machines

    Methods Enzymol.

    (2003)
  • J. Shen et al.

    Selective activation of cognate SNAREpins by Sec1/Munc18 proteins

    Cell

    (2007)
  • Y.S. Sou et al.

    Phosphatidylserine in addition to phosphatidylethanolamine is an in vitro target of the mammalian Atg8 modifiers, LC3, GABARAP, and GATE-16

    J. Biol. Chem.

    (2006)
  • M. Thumm et al.

    Isolation of autophagocytosis mutants of Saccharomyces cerevisiae

    FEBS Lett.

    (1994)
  • M. Tsukada et al.

    Isolation and characterization of autophagy-defective mutants of Saccharomyces cerevisiae

    FEBS Lett.

    (1993)
  • T. Weber et al.

    SNAREpins: minimal machinery for membrane fusion

    Cell

    (1998)
  • H. Weidberg et al.

    LC3 and GATE-16 N termini mediate membrane fusion processes required for autophagosome biogenesis

    Dev. Cell

    (2011)
  • H. Abeliovich et al.

    Dissection of autophagosome biogenesis into distinct nucleation and expansion steps

    J. Cell Biol.

    (2000)
  • E.L. Axe et al.

    Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum

    J. Cell Biol.

    (2008)
  • B. Brugger et al.

    Putative fusogenic activity of NSF is restricted to a lipid mixture whose coalescence is also triggered by other factors

    EMBO J.

    (2000)
  • C.Y. Chang et al.

    Atg19 mediates a dual interaction cargo sorting mechanism in selective autophagy

    Mol. Biol. Cell

    (2007)
  • L.V. Chernomordik et al.

    Protein-lipid interplay in fusion and fission of biological membranes

    Annu. Rev. Biochem.

    (2003)
  • F.S. Cohen et al.

    Parameters affecting the fusion of unilamellar phospholipid vesicles with planar bilayer membranes

    J. Cell Biol.

    (1984)
  • C. Dall’armi et al.

    The phospholipase D1 pathway modulates macroautophagy

    Nat. Commun.

    (2010)
  • T. Darsow et al.

    A multispecificity syntaxin homologue, Vam3p, essential for autophagic and biosynthetic protein transport to the vacuole

    J. Cell Biol.

    (1997)
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