Annexin A5 stimulates autophagy and inhibits endocytosis

Ghita Ghislat Carmen Aguado Erwin Knecht Laboratorio de Biologa Celular Centro de Investigaci on Prncipe Felipe CIBERER Avda. Autopista del Saler -Valencia Spain Author for correspondence () Summary Macroautophagy is a major lysosomal catabolic process activated particularly under starvation in eukaryotic cells. A new organelle, the autophagosome, engulfs cytoplasmic substrates, which are degraded after fusion with endosomes and/or lysosomes. During a shotgun proteome analysis of purified lysosomal membranes from mouse fibroblasts, a Ca2+-dependent phospholipid-binding protein, annexin A5, was found to increase on lysosomal membranes under starvation. This suggests a role for this protein, an abundant annexin with a still unknown intracellular function, in starvation-induced lysosomal degradation. Transient overexpression and silencing experiments showed that annexin A5 increased lysosomal protein degradation, and colocalisation experiments, based on GFP sensitivity to lysosomal acidic pH, indicated that this was mainly the result of inducing autophagosome-lysosome fusion. Annexin A5 also inhibited the endocytosis of a fluid-phase marker and cholera toxin, but not receptor-mediated endocytosis. Therefore, we propose a double and opposite role of annexin A5 in regulating the endocytic and autophagic pathways and the fusion of autophagosomes with lysosomes and endosomes. - Introduction In eukaryotic cells, macroautophagy (hereafter referred to as autophagy) is an important catabolic process for the clearance of intracellular components, including whole organelles, long-lived proteins and other cytosolic molecules (Knecht et al., 2009). The first step in autophagy involves the formation of a flat membrane sac (which in yeast is called the phagophore), the origin of which in mammalian cells is controversial. This structure surrounds cytoplasmic substrates and eventually closes, generating a double-membrane organelle, the autophagosome (Levine and Klionsky, 2004). The endoplasmic reticulum (ER) mainly (Axe et al., 2008; Hayashi-Nishino et al., 2009; Yla-Anttila et al., 2009), and to a lesser extent the mitochondria (Hailey et al., 2010; Mari et al., 2010) and plasma membrane (Ravikumar et al., 2010), appear to contribute proteins and lipids to the forming autophagosome under various situations (Cuervo, 2010). Lysosomes and/or late endosomes are the eventual target of autophagosomes for fusion events, to form autolysosomes containing lysosomal hydrolases that degrade the engulfed material and recycle the breakdown products. The power of yeast genetics has allowed to clarify several steps of the molecular mechanism of this process, and more than 30 evolutionarily conserved autophagy-related proteins have been identified by complementation screening (Klionsky et al., 2003). Some of these proteins are also present in mammalian cells (Knecht et al., 2009), although there are also mammalian-specific proteins involved in autophagy. Stress conditions, such as starvation, growth factor deprivation or protein aggregation upregulate autophagy to compensate for the lack of amino acids and energy and for other cell stresses and defects (Douglas and Dillin, 2010; Lum et al., 2005; Onodera and Ohsumi, 2005). However, mammalian cells sustain autophagy at a low basal level for the turnover of normally occurring misfolded proteins and damaged molecules and organelles. In mammalian cells, the main autophagy regulators are nutritional and hormonal (Meijer and Codogno, 2006). Thus, in early studies, insulin and amino acids were shown to inhibit autophagy, whereas glucagon activates it. In addition, glucose (which increases autophagy in mammalian cells, in contrast to yeast), vitamins, various growth factors and Ca2+ have been also implicated in autophagy regulation. Ca2+ is a universal messenger regulating many physiological functions in cells, such as secretion, contraction, metabolism, gene transcription and death, and it has been implicated also in some pathological processes (Berridge et al., 2003; Perez-Terzic et al., 1995). Regarding its role in autophagy, a pioneering study, exploring the contribution of Ca2+ to autophagy, established that stimulation of autophagy depends on its presence within intracellular Ca2+-storage compartments rather than on cytoplasmic Ca2+ (Gordon et al., 1993). More recently, it was reported that Ca2+ derived from extracellular sources and from ER is a signal for autophagy induction, on the basis of the activation of AMP-activated protein kinase by Ca2+/calmodulindependent kinase kinase-b (Hoyer-Hansen et al., 2007). However, other studies have provided evidence that, at least under certain conditions, an increase in cytosolic Ca2+ should inhibit autophagy (Williams et al., 2008). In addition to these conflicting conclusions on autophagy regulation, Ca2+ has a more well-established role in another closely related lysosomal function, endocytosis, where it is required for efficient fusion events between late endosomes and lysosomes or autophagosomes (Luzio et al., 2007; Pryor et al., 2000). While investigating the regulation of the lysosomal degradation of proteins, we carried out a two-dimensional differential gel electrophoresis (2D-DIGE) proteomic study of lysosomal membranes isolated from mouse fibroblasts, to identify proteins whose levels change under conditions of high or low proteolysis in the cells. The proteins on lysosomal membranes that increased under high proteolysis included at least three subunits of the vacuolar ATPase (Esteban et al., 2007) and three Ca2+-dependent phospholipid-binding proteins (annexin A1, annexin A5 and copine 1). In this study, we have concentrated on one of these Ca2+-binding proteins, annexin A5, because it is an abundant annexin with a still unknown intracellular function, and analysed its possible role in lysosomal protein degradation. Results Under starvation, annexin A5 translocates from the Golgi complex to lysosomal membranes in a Ca2+-dependent way To identify proteins involved in the regulation of lysosomal proteolytic pathways, we investigated, by 2D-DIGE and mass spectrometry analysis, lysosomal membranes isolated from e NIH3T3 cells incubated in KrebsHenseleit medium (KH) c without (starvation) and with insulin (I), amino acids (AA) or ien both (IAA). The level of the phospholipid-binding protein c annexin A5 was found, along with other proteins on the lS lysosomal membranes, to change under conditions that produce le high (KH), low (IAA) and intermediate (I or AA) proteolysis in C the cell (Fig. 1A). As shown in Fig. 1B, levels of annexin A5 in fo KH decreased after addition of AA or IAA, but not, apparently, la after addition of I. This was confirmed by western blot analysis rn of subcellular fractions enriched in nuclei, mitochondria and u lysosomes from NIH3T3 cells incubated under conditions that Jo produce high and low proteolysis. Annexin A5 was located in mitochondrial and lysosomal fractions, (...truncated)