Zinc-dependent lysosomal enlargement in TRPML1-deficient cells involves MTF-1 transcription factor and ZnT4 (Slc30a4) transporter

Abstract

Synopsis Zn is critical for a multitude of cellular processes, including gene expression, secretion and enzymatic activities. Cellular Zn is controlled by Zn-chelating proteins and by Zn transporters. The recent identification of Zn permeability of the lysosomal ion channel TRPML1, and the evidence of abnormal Zn levels in cells deficient in TRPML1, suggested a role for TRPML1 in Zn transport. Here we provide new evidence for such a role and identify additional cellular components responsible for it. In agreement with the previously published data, an acute siRNA-driven TRPML1 knockdown (KD) leads to the buildup of large cytoplasmic vesicles positive for Lysotracker and Zn staining, when cells are exposed to high concentrations of Zn. We now show that lysosomal enlargement and Zn buildup in TRPML1-KD cells exposed to Zn are ameliorated by KD of the Zn-sensitive transcription factor MTF-1 or Zn transporter ZnT4. TRPML1 KD is associated with a buildup of cytoplasmic Zn and with enhanced transcriptional response of mRNA for metallothionein 2a (MT2a). TRPML1 KD did not suppress lysosomal secretion, but it did delay Zn leak from the lysosomes into the cytoplasm. These data underscore a role for TRPML1 in Zn metabolism. Furthermore, they suggest that TRPML1 works in concert with ZnT4 to regulate Zn translocation between the cytoplasm and lysosomes.

A small molecule restores function to TRPML1 mutant isoforms responsible for mucolipidosis type IV

“…We used MLIV fibroblasts to verify these findings and to examine a possible rescue effect of MK6-83 on lysosomal zinc accumulation. We found that LysoTracker positive compartments of TRPML1 À / À fibroblasts displayed strong vesicular FluoZin-3 fluorescence when pretreated with ZnCl 2 as described previously 45,46 . The zinc accumulation in TRPML1 À / À fibroblasts was not reversible on treatment with MK6-83.…”

“…Different groups have previously reported that zinc accumulates in lysosomes in MLIV fibroblasts or after knockdown of TRPML1 44,45 . Heavy metal accumulation may ultimately lead to the neurodegenerative effects seen in MLIV patients.…”

Zinc efflux through lysosomal exocytosis prevents zinc-induced toxicity

“…The resulting cytoplasmic Zn 2+ spikes were measured using livecell confocal microscopy and FluoZin-3,AM as described previously (Kukic et al, 2013) Fig. 1A shows that the exposure of Baf-treated cells to Zn 2+ caused a significantly higher FluoZin-3,AM response than the exposure of untreated cells to Zn 2+ .…”

“…It is tempting to speculate that Zn 2+ export into organelles serves as a first line of defense to provide temporary Zn 2+ storage, giving cells time to upregulate Zn 2+ chelators and transporters. Our recent data on the role of lysosomes in Zn 2+ handling, as well as some recently published results suggest that lysosomes play a role as such Zn 2+ sinks, temporarily storing Zn 2+ (Hwang et al, 2008;Kukic et al, 2013). In this paper, we sought to delineate the role of lysosomes in protection against Zn 2+ -induced toxicity.…”

“…Alternatively, it is possible that lysosomal Zn 2+ dissipates and lysosomes constitute only a temporary Zn 2+ storage site. Our recently published data suggest that the lysosomal ion channel transient receptor potential mucolipin 1 (TRPML1, also known as mucolipin1, encoded by the MCOLN1 gene) is at least partly responsible for dissipating lysosomal Zn 2+ into the cytoplasm (Kukic et al, 2013). It should be noted that lysosomes fuse with the plasma membrane through a process involving a specific SNARE complex, which includes the VAMP7 protein and synaptotagmin VII (SYT7) (Martinez-Arca et al, 2000;Braun et al, 2004;Rao et al, 2004;Logan et al, 2006;Mollinedo et al, 2006).…”