Golgi-resident Small GTPase Rab33B Interacts with Atg16L and Modulates Autophagosome Formation

Abstract

Macroautophagy is a mechanism of degradation of cytoplasmic components in all eukaryotic cells. In macroautophagy, cytoplasmic components are wrapped by double-membrane structures called autophagosomes, whose formation involves unique membrane dynamics, i.e., de novo formation of a double-membrane sac called the isolation membrane and its elongation. However, the precise regulatory mechanism of isolation membrane formation and elongation remains unknown. In this study, we showed that Golgi-resident small GTPase Rab33B (and Rab33A) specifically interacts with Atg16L, an essential factor in isolation membrane formation, in a guanosine triphosphate-dependent manner. Expression of a GTPase-deficient mutant Rab33B (Rab33B-Q92L) induced the lipidation of LC3, which is an essential process in autophagosome formation, even under nutrient-rich conditions, and attenuated macroautophagy, as judged by the degradation of p62/sequestosome 1. In addition, overexpression of the Rab33B binding domain of Atg16L suppressed autophagosome formation. Our findings suggest that Rab33 modulates autophagosome formation through interaction with Atg16L. INTRODUCTIONMacroautophagy is a well-conserved mechanism in all eukaryotic cells, and it is important for cells to rid the cell of injured or unwanted cytoplasmic material and to degrade normal cellular components in response to energy needs (Levine and Klionsky, 2004). Macroautophagy (simply referred to as autophagy hereafter) is thought to be the major autophagic pathway, and functions not only as a nutrient supply but also as a defense against stresses, including bacterial intrusion, unfolded protein accumulation, and so on (Mizushima, 2007).In mammalian autophagy, a precursor structure, a crescent-shaped small membrane compartment called isolation membrane core, is present in the initial step in autophagosome formation and it elongates to form characteristic double-membrane structures, called autophagosomes. Finally, autophagosomes fuse with lysosomes to degrade its contents (Yoshimori, 2004).During the past decade, many genes essential for autophagy (i.e., ATG genes) have been identified by genetic analysis of Saccharomyces cerevisiae (Thumm et al., 1994;Tsukada and Ohsumi, 1993; reviewed in Klionsky et al., 2003), and because most of the yeast ATG genes have mammalian counterparts, the fundamental molecular machinery (or proteinprotein interaction cascade) for autophagy is thought to be conserved in all eukaryotic cells (Ohsumi, 2001;Mizushima et al., 2002).Atg5 interacts with Atg12, a ubiquitin-like protein (Ubl) covalently conjugated to Atg5, and with Atg16L, and these proteins form ϳ800-kDa complex through homooligomerization of Atg16L (referred to as Atg5-12/16L complex hereafter). The complex is specifically present on isolation membranes and never present on mature autophagosomes (Mizushima et al., 1998(Mizushima et al., , 2001(Mizushima et al., , 2003. LC3, another Ubl, is conjugated to phosphatidylethanolamine (PE) and localized at elongating isolation membranes and mature aut...

Overview of macroautophagy regulation in mammalian cells

“…The location of the Atg16L complex at the phagophore dictates the location of the LC3-conjugation reaction [35]. It has been suggested that the localization of the Atg16L complex on the membrane may be dependent on the monomeric GTPase Rab33B [36]. Interestingly, another Rab protein, Rab5, has been shown to regulate the conjugation of Atg12 at the phagophore through hVsp34 [37].…”

Vesicular trafficking and autophagosome formation

“…63 It was shown that the Golgi residents (Rab33a and Rab33b) bind directly to Atg16L in a GTP-dependent manner and overexpression of GFP-Rab33b causes Atg16L recruitment to the Golgi. A constitutively active mutant of Rab33b (Rab33b-Q92L) significantly increased LC3-II levels regardless of the nutrient state, but did not lead to formation of LC3-positive puncta, normally found under starvation conditions.…”

Direct link between Atg protein and small GTPase Rab: Atg16L functions as a potential Rab33 effector in mammals

“…12 and unpublished data). Of the 60 mammalian Rab isoforms tested, three Atg proteins, Atg5, Atg12, and Atg16L, all of which are essential for isolation membrane formation, were co-purified with the GTP-bound form of Rab33B, 12 previously reported as a cis-Golgi-resident Rab. 13 These Atg proteins form a tight complex (referred to as the Atg12-5/16L complex below), and the complex is known to be essential for isolation membrane formation.…”

“…[19][20][21] In this scenario, Rab33 should be an essential factor for autophagosome formation in mammalian cells. Actually, overexpression of the Rab33-binding domain of Atg16L strongly suppresses starvation-induced autophagy (i.e., there is a positive correlation between the Rab33B-binding activity and autophagy-inhibiting activity of Atg16L), 12,17 suggesting that the CC domain of Atg16L traps endogenous Rab33 molecules. However, small interfering RNA-mediated knockdown of endogenous Rab33B has little effect on autophagosome formation.…”