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Zinc is essential for all organisms and yet detrimental at elevated levels. Hence, homeostasis of this metal is tightly regulated. The Zrt/Irt-like proteins (ZIPs) represent the only zinc importers in metazoans. Mutations in human ZIPs cause serious disorders, but the mechanism by which ZIPs transfer zinc remains elusive. Hitherto, structural information is only available for a model member, BbZIP, and as a single, ion-bound conformation, precluding mechanistic insights. Here, we elucidate an inward-open metal-free BbZIP structure, differing substantially in the relative positions of the two separate domains of ZIPs. With accompanying coevolutional analyses, mutagenesis, and uptake assays, the data point to an elevator-type transport mechanism, likely shared within the ZIP family, unifying earlier functional data. Moreover, the structure reveals a previously unknown ninth transmembrane segment that is important for activity in vivo. Our findings outline the mechanistic principles governing ZIP-protein transport and enhance the molecular understanding of ZIP-related disorders.

Aquaporins (AQPs) are membrane-bound water channels that play crucial roles in maintaining the water homeostasis of the human body. Here, we present a protocol for high-yield recombinant expression of human AQPs in the methylotropic yeast

Aquaporins (AQPs) are water channels embedded in the cell membrane that are critical in maintaining water homeostasis. We describe a protocol for determining the water permeation capacity of AQPs reconstituted into proteoliposomes. Using a stopped-flow setup, AQP embedded in proteoliposomes are exposed to an osmogenic gradient that triggers water flux. The consequent effects on proteoliposome size can be tracked using the fluorescence of an internalized fluorophore. This enables controlled characterization of water flux by AQPs. For complete details on the use and execution of this protocol, please refer to Kitchen et al. (2020). [Abstract copyright: © 2022 The Authors.]