br The inhibitory effect of ATP

The inhibitory effect of ATP7B knockdown on lysosomal exocytosis is likely mediated by the resulting oxidative stress induced by Cu, as exposure to oxidative stress induced by tert-Butyl hydroperoxide (TBHP) inhibited β-hex exocytosis as well, indicating that oxidative stress inhibits lysosomal exocytosis (Fig. 3D). Based on these results we propose that Cu stimulates lysosomal exocytosis to accelerate Cu extraction and prevent oxidative damage. Lysosomes have emerged as key determinants of transition metal detoxification as lysosomal exocytosis was shown to be indispensable for removal of Cu and Zn from cells. Although the molecular determinants of metal excretion via lysosomal exocytosis have been delineated [3], [4], the functional relations between transition metals and exocytosis are not well understood. The active response of lysosomal metal transporters to transition metal exposure (increased transcription and translocation) suggests sophisticated relationship between transporters and lysosomal exocytosis. We show that exposure to Cu stimulates lysosomal exocytosis. The aspects of the lysosomal exocytosis stimulated by Cu require Ca. While Ca release through the lysosomal ion channel TRPML1 was suggested to drive lysosomal exocytosis, it is unlikely to contribute to the Cu-dependent lysosomal exocytosis. First, TRPML1 does not conduct Cu and does not seem to be activated by Cu [17]. Second, siRNA-driven TRPML1 knockdown described in our previous studies did not affect the Cu-dependent component of the lysosomal exocytosis (not shown). Finally, stimulation of the lysosomal exocytosis by Cu is inhibited by removal of extracellular Ca (Fig. 2C) or by addition of extracellular LaCl3 (Fig. 2D), suggesting involvement of a plasma membrane Ca channel activated by Cu and poorly sensitive to La. Information on the effect of Cu on plasma membrane gsk3 inhibitor is limited (see summary in a recent review [12]). TRPA1 and some members of the TRPM family are among candidates for the role of such a channel. On the other hand, the fact that we were unable to detect any measurable spike in cytoplasmic Ca in response to the extracellular Cu application suggests a possibility of Cu inducing a very local Ca influx, or an effect on La-insensitive Ca transporter. Finally, it is possible that Cu sensitizes the machinery responsible for the lysosomal fusion with the plasma membrane to Ca. We find that the Cu-dependent component of the lysosomal exocytosis persists in ATP7B-deficient cells (Fig. 3B), making it unlikely that Cu exerts its effect via ATP7B-dynactin interaction only. However, the stimulation of lysosomal exocytosis by Cu was absent in VAMP7-depeleted cells (Fig. 2A), again underscoring the possible role of Cu in Ca-dependent aspects of the lysosomal exocytosis. Whether or not Cu regulates the Ca-dependence of other components of the membrane fusion machinery remains to be answered. The effect of ATP7B knockdown on lysosomal exocytosis is intriguing. ATP7B not only mediates lysosomal Cu uptake; it also facilitates the transport of lysosomes to the plasma membrane [3]. The reduction of lysosomal exocytosis in control cells transfected with ATP7B siRNA (Fig. 1C) can be a consequence of the latter. Furthermore, reducing ATP7B protein levels affects Cu homeostasis in two ways: lysosomal Cu uptake is reduced and evacuation of lysosomal Cu is prevented. Under these circumstances even low levels of Cu present in growth medium can induce oxidative stress and further affect lysosomal exocytosis. Taken together with the increased oxidative stress in ATP7B-knockdown cells (Fig. 3B) and with the suppression of the lysosomal exocytosis by oxidative stress, it provides more evidence for a dynamically regulated cytoprotective system driven by lysosomal uptake of transition metal followed by their exocytosis. Beyond their role in digestion, this redefines lysosomes as cytoprotective organelles.