New Insights into the Mechanisms of Macroautophagy in Mammalian Cells

Abstract

Macroautophagy is a self-digesting pathway responsible for the removal of long-lived proteins and organelles by the lysosomal compartment. Parts of the cytoplasm are first segregated in double-membrane-bound autophagosomes, which then undergo a multistep maturation process including fusion with endosomes and lysosomes. The segregated cytoplasm is then degraded by the lysosomal hydrolases. The discovery of ATG genes has greatly enhanced our understanding of the mechanisms of this pathway. Two novel ubiquitin-like protein conjugation systems were shown to function during autophagosome formation. Autophagy has been shown to play a role in a wide variety of physiological processes including energy metabolism, organelle turnover, growth regulation, and aging. Impaired autophagy can lead to diseases such as cardiomyopathy and cancer. This review summarizes current knowledge about the formation and maturation of autophagosomes, the role of macroautophagy in various physiological and pathological conditions, and the signaling pathways that regulate this process in mammalian cells.

Introduction

Autophagy is an evolutionarily conserved and strictly regulated lysosomal pathway that degrades cytoplasmic material and organelles. This pathway is activated under stress conditions such as amino acid starvation, unfolded protein response, or viral infection. Depending on the delivery route of the cytoplasmic material to the lysosomal lumen, four different autophagic routes are known: (1) macroautophagy, or simply autophagy, (2) microautophagy, (3) chaperone-mediated autophagy, and (4) crinophagy (Fig. 5.1). In macroautophagy, a portion of cytoplasm to be degraded is first wrapped inside a specialized organelle, the autophagosome, which then fuses with lysosomal vesicles and delivers the engulfed cytoplasm for degradation (Arstila and Trump, 1968) (Fig. 5.2). In microautophagy, the lysosomal membrane itself sequesters a portion of cytoplasm by a process that resembles pinching off of phagosomes or pinosomes from the plasma membrane (Ahlberg et al., 1982). Starvation-induced macroautophagic uptake of cytoplasmic material appears to be a nonselective process (Kopitz et al., 1990); organelles are sequestered at the same frequency as they exist in the cytoplasm. In chaperone-mediated autophagy, proteins possessing a specific sequence signal are transported from the cytoplasm, through the lysosomal membrane, to the lysosomal lumen (Cuervo and Dice, 1996). The lysosome-associated membrane protein (LAMP)-2 was proposed to act as a receptor in chaperone-mediated autophagy. A fourth autophagic route, crinophagy, has also been described (Glaumann, 1989). In crinophagy, secretory vesicles directly fuse with lysosomes, which leads to degradation of the granule contents. This review concentrates on the (macro)autophagic pathway in mammalian cells.

After induction by a stress signal such as amino acid starvation, the first step in (macro)autophagy is the formation of an autophagosome (Fig. 5.2). A flat membrane cistern elongates and wraps itself around a portion of cytoplasm, forming a double-membrane-bound autophagosome. This membrane cistern has been called the phagophore, or isolation membrane. Autophagosomes next receive lysosomal constituents, such as lysosomal membrane proteins and proton pumps, from endosomal vesicles via vesicle-mediated transport, and/or by fusion with late endosomes or multivesicular bodies (MVBs). Finally, the limiting membranes of autophagosomes fuse with the limiting membranes of lysosomes (Berg 1998, Dunn 1990b, Gordon 1992, Lawrence 1992, Liou 1997, Punnonen 1993, Tooze 1990) (Fig. 5.2). In this process, the cytoplasm, still engulfed by the inner limiting membrane, is delivered to the endo/lysosomal lumen (Figure 5.2, Figure 5.3). Both the cytoplasm and the membrane around it are then degraded by lysosomal hydrolases, and the degradation products are transported back to cytoplasm, where they can be reused for metabolism. In yeast cells, autophagosomes directly fuse with the vacuole, the counterpart of the mammalian lysosome, without an endosomal fusion step (Baba et al., 1994).

By definition, autophagosomes, also called initial autophagic vacuoles (AVi), do not yet contain lysosomal membrane proteins or enzymes, and are not acidic (Dunn, 1990a). During the maturation process, autophagosomes develop into late, or degradative autophagic vacuoles (AVd), which are acidic and contain lysosomal membrane proteins and enzymes (Dunn, 1990b) (Fig. 5.3). After fusion with lysosomes, autophagosomes are called autolysosomes. Quantitative immunoelectron microscopy has been used to demonstrate the enrichment of lysosomal membrane proteins and enzymes in late autophagic vacuoles/autolysosomes (Eskelinen 2002a, Tanaka 2000).

The lack of integral membrane proteins in autophagosomes (AVi) was first revealed by freeze–fracture electron microscopy. Other cellular membranes such as lysosomal and endoplasmic reticulum membranes contain numerous integral membrane particles, considered to represent integral membrane protein molecules revealed by the freeze–fracture procedure. However, the surfaces of the membranes limiting autophagosomes are almost completely smooth (Fengsrud 2000, Punnonen 1989, Rez 1980).

The origin of the membrane cistern forming new autophagosomes has been the subject of numerous studies, but still this issue is unresolved in mammalian cells. Many older ultrastructural studies suggested that smooth endoplasmic reticulum (ER) cisternae are the source of autophagosome membranes (Dunn, 1994), but evidence against this interpretation has also been published (Yamamoto et al., 1990). Studies in yeast have revealed that autophagosomes originate from a unique compartment called the preautophagosomal structure (PAS) (Kim 2002, Suzuki 2007). Because the autophagic pathway and genes involved in it are well conserved from yeast to mammals, it is possible that a similar or equivalent unique compartment is the source of membrane in mammalian cells.

Interestingly, ER cisternae are often observed to closely surround nascent autophagosome membranes (Fig. 5.4). In yeast, membrane transport out of the ER seems to contribute to autophagosome formation. Coat protein complex II (COPII)-coated vesicles transport material from the ER to the Golgi complex. Yeast mutants defective in the Sec23–Sec24 subcomplex of COPII vesicles are unable to form autophagosomes. However, mutants defective in Sec12 or the Sec23–Sec31 subcomplex (also needed for COPII vesicle formation) have no autophagy defects (Hamasaki 2003, Ishihara 2001). Trs85, a component of the TRAPP complexes, is also required for autophagy in yeast (Meiling-Wesse 2005, Nazarko 2005). The transport protein particle, or TRAPP, is a complex of 10 subunits that is essential for tethering of ER-derived transport vesicles to Golgi membranes. Further, conventional membrane fusion machinery, including N-ethylmaleimide-sensitive fusion protein [or N-ethylmaleimide-sensitive factor (NSF)], soluble NSF attachment protein (SNAP), and SNAP receptors, are not needed for autophagosome formation in yeast (Ishihara 2001, Suzuki 2007). These results suggest that unconventional membrane traffic pathways are used during autophagosome formation.

Starvation-induced macroautophagic uptake of cytoplasmic material has been considered a nonselective process (Kopitz et al., 1990). However, selective autophagic uptake of peroxisomes (pexophagy) has been described in yeast (Bellu 2003, Hutchins 1999). Also in mammalian cells, autophagy seems to be necessary for the removal of excess peroxisomes (Iwata et al., 2006), but this uptake is not as strictly selective for peroxisomes as pexophagy in yeast. A selective sequestration of the endoplasmic reticulum was described during the unfolded protein response in yeast (Bernales et al., 2006). Unfolded protein response triggers autophagy also in mammalian cells (Ogata 2006, Yorimitsu 2006) but this uptake does not seem to be strictly selective for the ER, although it is possible that the ER is enriched in autophagosome contents. There is also evidence for a selective uptake of mitochondria by macroautophagy in mammalian hepatocytes (Elmore 2001, Kim 2007). This selective uptake has been called mitophagy and was suggested to be important for the removal of dysfunctional mitochondria. The mechanism of this proposed selectivity is currently unknown. It is, however, possible that reactive oxygen species (ROS) might play a role in the recognition of mitochondria for segregation (Scherz-Shouval et al., 2007) (see Section 4.1.2).