Lipid transfer proteins (LTPs) maintain the specialized lipid compositions of organellar membranes ^1,2 . In humans, many LTPs are implicated in diseases ³ , but the cargo and auxiliary lipids that facilitate the transfer of the majority of LTPs remain unknown. Here we combined biochemical, lipidomic and computational methods to systematically characterize LTP–lipid complexes ⁴ and measure how LTP gains of function affect cellular lipidomes. We identified bound lipids for around half of the hundreds of LTPs that we analysed, confirming known ligands and identifying new ones across most LTP families. Gains in LTP function affected the cellular abundance of both their known and newly identified lipid ligands, indicating comparable functional relevance of the two ligand sets. Using structural bioinformatics, we characterized mechanisms that contribute to lipid selectivity and identified preferences based on headgroup or acyl chain. We demonstrate some basic principles of how LTPs mobilize their ligands. They commonly interact with several classes of lipids and exhibit broad but selective preference for particular headgroups and for lipid species with shorter acyl chains that contain one or two unsaturated carbons, suggesting that only subsets of lipid species are efficiently mobilized. The datasets represent a resource for further analysis in different cell types and states, such as those associated with pathologies.

Nitrification is a major process in the global nitrogen cycle, initiated by the oxidation of ammonia to nitrite. The ammonia-oxidising bacterium Nitrosomonas europaea catalyses this first and rate-limiting step and depends on ammonium both as an energy substrate and as a nitrogen source, making ammonium acquisition central to its physiology. Cellular ammonium transport is mediated by the Amt/Mep/Rh superfamily, whose members share a conserved fold despite divergent physiological roles. Remarkably, N. europaea lacks a canonical prokaryotic Amt transporter and instead expresses NeRh50, a Rh-family protein proposed to have been acquired from a eukaryotic lineage. Here, we determine a cryo-EM structure of NeRh50 that reveals tightly associated lipids at the monomer–monomer interfaces, including density consistent with an unusual inverted lipid orientation. Native mass spectrometry and molecular dynamics simulations further support persistent lipid association at these interfaces. NeRh50 also differs functionally from canonical AmtB, selectively mediating electrogenic ammonium transport with ∼70-fold lower apparent affinity. Transport is enhanced under alkaline conditions but not by an imposed proton gradient, suggesting membrane-potential dependence. Together, these findings reveal divergence from canonical Amt proteins in both membrane-associated architecture and transport properties, providing insight into the functional and evolutionary diversification of the Amt/Mep/Rh superfamily.