Abstract
Endolysosomes are key players in cell physiology, including molecular exchange, immunity, and environmental adaptation. They are the molecular targets of some pore-forming aerolysin-like proteins (ALPs) that are widely distributed in animals and plants and are functionally related to bacterial toxin aerolysins. βγ-CAT is a complex of an ALP (BmALP1) and a trefoil factor (BmTFF3) in the firebelly toad (Bombina maxima). It is the first example of a secreted endogenous pore-forming protein that modulates the biochemical properties of endolysosomes by inducing pore formation in these intracellular vesicles. Here, using a large array of biochemical and cell biology methods, we report the identification of BmALP3, a paralog of BmALP1 that lacks membrane pore-forming capacity. We noted that both BmALP3 and BmALP1 contain a conserved cysteine in their C-terminal regions. BmALP3 was readily oxidized to a disulfide bond-linked homodimer, and this homodimer then oxidized BmALP1 via disulfide bond exchange, resulting in the dissociation of βγ-CAT subunits and the elimination of biological activity. Consistent with its behavior in vitro, BmALP3 sensed environmental oxygen tension in vivo, leading to modulation of βγ-CAT activity. Interestingly, we found that this C-terminal cysteine site is well conserved in numerous vertebrate ALPs. These findings uncover the existence of a regulatory ALP (BmALP3) that modulates the activity of an active ALP (BmALP1) in a redox-dependent manner, a property that differs from those of bacterial toxin aerolysins.
Highlights
Cellular membranes are essential for defining the border and ensuring the function of all living cells [1, 2]
Sequence alignment revealed that the protein shared 27% sequence identity with the bgCAT BmALP1 subunit, suggesting that it was homologous to BmALP1, and we termed it BmALP3
Further sequence analysis revealed that BmALP3 contained only the aerolysin domain; this is different from BmALP1, which contained the bg-crystallin domain and aerolysin domain (Fig. 1B and Fig. S1B)
Summary
Cellular membranes are essential for defining the border and ensuring the function of all living cells [1, 2]. A coimmunoprecipitation assay was performed and showed a significant interaction between BmALP3 and bg-CAT in toad skin secretions (Fig. 1E).
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