Regulation of ion channel and transporter activity is necessary to preserve cellular and organellar homeostasis. Among the various regulatory mechanisms, including posttranslational modifications, second messengers, and feedback loops, auxiliary β-subunits play a particularly important role. Within the CLC family of chloride channels and transporters, several members associate with β-subunits, which modulate their stability, localization, and function.
In this work, we identified and characterized TMEM9 and TMEM9B proteins as obligatory β-subunits of ClC-3, ClC-4, and ClC-5 chloride/proton exchangers. We demonstrated that these proteins form complexes with vesicular CLCs and are mutually required for protein stability in vivo. Despite earlier reports suggesting predominantly lysosomal localization of both TMEM9 isoforms, we showed that TMEM9 proteins are instead located in the same vesicular compartments as the CLCs they interact with.
Functionally, both TMEM9A and TMEM9B regulate the intracellular trafficking of the CLCs and, importantly, they strongly suppress CLC-mediated ion transport. Fine-tuned control of the transporters’ activity is necessary, as evidenced by loss-of-function or gain-of-function mutations in CLCs associated with various diseases. Thus, we dissected key regulatory motifs localized in the C-termini of TMEM9 proteins, which are essential for both proper localization and function of CLC/TMEM9 complexes. Specifically, we demonstrated that the phosphorylated acidic cluster is necessary to properly regulate the endocytosis and recycling of CLC/TMEM9 complexes, while the C-terminal inhibitory domain is needed to inhibit the ion transport mediated by the respective CLCs. Disruption of the TMEM9-mediated inhibition leads to a pathological increase of ClC-3–5 ion transport activity. Moreover, we suggested the possible relevance of phosphorylation for both trafficking and inhibitory roles of TMEM9 proteins.
Taken together, our findings establish TMEM9 and TMEM9B as obligatory β-subunits of ClC-3, ClC-4, and ClC-5, which regulate both their localization and function. This regulation may be dynamically controlled by phosphorylation of TMEM9 proteins, and its disruption contributes to the disease-related overactivity of transporters.
