Chapter 1 Biochemical Methods to Monitor Autophagy‐Related Processes in Yeast
Introduction
Macroautophagy (hereafter autophagy) is one of the major cellular catabolic processes and is evolutionarily conserved in eukaryotic cells from yeast to human (Klionsky, 2005, Reggiori and Klionsky, 2002). This process occurs constitutively at basal levels but is dramatically stimulated by various extra‐ and intracellular stresses, such as nutrient or growth factor limitation, oxidative stress, and accumulation of damaged organelles or misfolded proteins (Mizushima and Klionsky, 2007).
During autophagy, cytoplasmic constituents including entire organelles are sequestered within double‐membrane vesicles, termed autophagosomes. The completed autophagosomes are then transported to degradative compartments (e.g., vacuoles or lysosomes). The inner vesicles, named autophagic bodies, are released into the vacuole lumen (or exposed to the lumen of the lysosome, which is too small to encompass the vesicle) and broken down through hydrolysis (Klionsky, 2005, Xie and Klionsky, 2007). After the cargo has been degraded in the lysosome/vacuole, the breakdown products are exported to the cytosol for reuse (Yang et al., 2006).
Autophagy is generally considered a nonspecific degradative process. Various studies have reported that autophagy and related processes (all of which are grouped under the term autophagy‐related) have multiple functions in cell physiology. In particular, autophagy‐related processes play critical roles in cellular remodeling through degradation, thus affecting normal developmental stages (Levine and Klionsky, 2004). Autophagy is are also involved in various pathophysiological conditions such as cancer, pathogen invasion, and certain types of neurodegeneration (Mizushima and Klionsky, 2007, Mizushima, 2008, Rubinsztein, 2005).
Most of the Atg proteins that are involved in different types of autophagy have been characterized by molecular genetic studies using fungal systems (Harding, 1995, Thumm, 1994, Titorenko, 1995, Tsukada and Ohsumi, 1993). Up to now, 31 Atg proteins have been identified in various fungi. The functions of these proteins are involved in distinct steps of autophagy‐related pathways including induction, vesicle nucleation and expansion, retrieval of Atg proteins from the forming vesicle, breakdown of the autophagic body and efflux of the degradation products. However, the exact functions of these components are still being actively investigated.
A subset of proteins including the Atg1 kinase form complexes that play roles in various steps of the autophagy‐related pathways. The Atg1 complexes are particular candidates for regulating autophagy induction, which may be mediated directly or indirectly by Tor kinase (Kamada et al., 2000). Most Atg proteins in yeast reside transiently at the phagophore assembly site (PAS), which is considered the potential nucleating site for sequestering vesicle formation, and a subgroup of Atg1 complexes has a critical role in initiating the PAS recruitment of other Atg proteins that function during nonspecific (starvation‐induced) autophagy (Cheong et al., 2008). On the other hand, Atg11, which is an Atg1‐interacting protein, is important for PAS assembly to generate the sequestering vesicles that are used for the biosynthetic autophagy‐related cytoplasm to vacuole targeting (Cvt) pathway that operates under vegetative conditions (Shintani and Klionsky, 2004).
In addition to the Atg1 complexes, most of the Atg proteins can be separated into three functional subgroups. (1) Atg9 is a transmembrane protein that may be involved in delivering membrane to the expanding sequestering vesicle. Atg9 appears to cycle between the PAS and non‐PAS pools; the latter may represent the source of the membrane(s) that contribute to autophagosome and Cvt vesicle formation. Atg1, Atg13, Atg2, and Atg18 are required for retrograde movement of Atg9 away from the PAS and back to the peripheral sites; Atg23 and Atg27 (and Atg11, which acts at multiple steps) are involved in anterograde movement (He, 2006, Reggiori, 2004, Reggiori, 2005). (2) The sole yeast phosphatidylinositol 3‐kinase (PtdIns3K), Vps34, and its modulating proteins have multiple functions for vesicular transport (Kihara et al., 2001). The core components include Vps34, Vps15, and Atg6/Vps30, which play a role in vesicle formation both for autophagy and the vacuolar protein sorting (Vps) pathway (Kihara, 2001, Stack, 1993). In addition to these components, two different proteins specify the function of each PtdIns3K complex within their respective pathways; Atg14 directs PtdIns3K to function in autophagy‐related processess and Vps38 directs the complex into the Vps pathway. (3) Two ubiquitin‐like (Ubl) proteins, Atg12 and Atg8, are involved in autophagy‐related mechanisms (George, 2000, Huang, 2000, Ichimura, 2000, Kirisako, 2000, Mizushima, 1998). These proteins are conjugated to Atg5 and the lipid phosphatidylethanolamine (PE), respectively. Atg8 is initially synthesized with a C‐terminal arginine residue that is proteolytically removed through the action of Atg4. The E1‐like activating enzyme Atg7 activates both Atg12 and Atg8, then transfers them to E2‐like conjugating enzymes, Atg10 and Atg3, which attach them to their respective targets. The Atg12–Atg5 conjugate binds to Atg16 to form a tetrameric structure of Atg12–Atg5‐Atg16 (Kuma et al., 2002). Recent data indicate that the Atg12–Atg5‐Atg16 complex acts as an E3‐like enzyme for conjugation of Atg8 to PE, although conjugation can occur in the absence of these proteins. The Atg12–Atg5 conjugation appears to be irreversible, whereas Atg8–PE is subsequently deconjugated through a second Atg4‐dependent cleavage (Kirisako et al., 2000) and the liberated Atg8 is presumably reused for another round of conjugation.
Next, additional proteins such as SNAREs and Rab GTPases play a role in the fusion of autophagosomes and Cvt vesicles with the vacuole, but these proteins are common to all other vacuolar fusion events and thus are not considered specific Atg proteins (Gutierrez, 2004, Klionsky, 2005). The membrane of the autophagic/Cvt bodies within the vacuolar lumen is lysed in an Atg15‐dependent manner, allowing access to the cargo; Atg15 is a putative yeast lipase (Epple, 2001, Teter, 2001). Most autophagic cargo molecules are broken down by vacuolar hydrolases. Subsequently, Atg22 and other permeases allow efflux of the resulting macromolecules into the cytosol for recycling (Yang et al., 2006).
As indicated previously, autophagy is generally considered a nonspecific process for the bulk degradation of cytoplasmic constituents, but various types of specific autophagy‐related processes have been reported, including the Cvt pathway. Another well‐characterized example is the degradation of peroxisomes in methylotrophic yeasts; shifting cells from conditions where peroxisomes are needed to one in which they are superfluous results in their specific degradation to adapt to the changing metabolic demands of the cell (Monastyrska and Klionsky, 2006). In addition, damaged organelles such as mitochondria might be removed by selective autophagy (Kiššová, 2007, Zhang, 2007). See the chapters by Camougrand et al., Oku and Sakai, and van Zutphen et al. in this volume for detailed information on specific organelle degradation.
The cytoplasm‐to‐vacuole‐targeting (Cvt) pathway is a unique example of selective autophagy in fungi, because it is the only known biosynthetic autophagy‐like pathway. As cargo proteins, the oligomeric form of the vacuolar hydrolase α‐mannosidase (Ams1) and precursor aminopeptidase I (prApe1) are sequestered by double‐membrane vesicles and transported from the cytosol to the vacuole through this alternative (i.e., not through a portion of the secretory pathway as occurs with most resident vacuolar hydrolases) targeting route that extensively overlaps with bulk autophagy (Hutchins and Klionsky, 2001, Klionsky, 1992).
Although these various autophagy‐related processes mostly share the same Atg protein machinery, there are clear morphological and mechanistic differences between specific and nonspecific pathways. For example, the Cvt pathway occurs constitutively during growth in nutrient‐rich conditions. The main cargo, prApe1 and Ams1, are sequestered within double‐membrane vesicles; Cvt vesicles are approximately 150 nm in diameter and appear to exclude bulk cytosol (Baba, 1997, Hutchins and Klionsky, 2001). In contrast, nonspecific autophagy is usually induced by starvation but also occurs at a basal level under vegetative conditions. Autophagosomes are relatively larger than Cvt vesicles, approximately 300–900 nm in diameter and include bulk cytosol and even entire organelles (Baba et al., 1994).
Accordingly, each pathway shows unique features, which suggests that certain types of Atg proteins may have critical roles to differentiate one pathway from another. For example, the adapter protein Atg11 determines the cargo selectivity for various types of specific autophagy‐related pathways (Shintani et al., 2002), whereas an autophagy‐specific protein, Atg17, has a role in determining the magnitude of the nonspecific autophagic response, which might allow the cell to initiate the uptake of bulk cytoplasm when shifting to autophagy‐inducing conditions (Cheong et al., 2005).
To understand the nature of different types of autophagy‐related pathways at the molecular level, a significant number of assays have been developed in various eukaryotic systems, including yeast (Klionsky et al., 2007). Autophagosomes were first identified by electron microscopy (EM) based on the unique double‐membrane morphology of the vesicle in mammalian cells. In yeast, the identification of the Cvt pathway that occurred concomitant with the analysis of autophagosomes by EM provided an extremely useful marker protein, precursor aminopeptidase I, for analysis of selective autophagy (Harding, 1996, Klionsky, 1992, Takeshige, 1992). Examination of this protein has provided a means to generate quantitative molecular and biochemical information that provides insight into the specific functions of Atg proteins at individual steps of autophagy‐related processes.
In this chapter we describe several conventional assays in baker's yeast to monitor certain types of selective and nonselective autophagy that rely on the detection of different marker proteins.
Section snippets
Background
Precursor Ape1 is a specific cargo of Cvt vesicles or autophagosomes, depending on the nutrient conditions. In contrast to most vacuolar proteins, prApe1 does not have a signal sequence to allow for translocation into the endoplasmic reticulum (ER) (Klionsky et al., 1992). After synthesis in the cytosol, prApe1 rapidly oligomerizes to become a dodecamer and further assembles into a higher‐order oligomeric structure, which is called the Ape1 complex (Oda et al., 1996) (Fig. 1.1). This complex is
Background
In addition to the Cvt pathway, another type of selective autophagy is the degradation of excess peroxisomes, termed pexophagy, which is mostly studied in various fungi (Dunn et al., 2005). When fungi grow on carbon sources that require peroxisome function, such as oleic acid (Saccharomyces cerevisiae is not methylotrophic) or methanol (for Pichia pastoris or Hansenula polymorpha), the peroxisomes are proliferated. Then, if a preferred carbon source such as glucose is added to the medium, the
High‐efficiency yeast transformation
This protocol is based on Gietz and Woods (2002)
- 1
S. cerevisiae strains are grown in DeLong culture flasks overnight at 30 °C in YPD. Grow approximately 1.5 ml of cell culture per transformation. On the next day, the cultured cells are diluted to O.D600 = 0.2 in fresh YPD and regrown to mid‐log phase (O.D600 = 0.8–1).
- 2
Sufficient cells for the desired number of transformations (1–1.5 O.D.600 units per transformation) are harvested by centrifugation at 5,000×g for 3 min.
- 3
Harvested cells are washed in
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