Breaking the annotation barrier: An initial subcellular localization atlas of Giardia's hypothetical and conserved hypothetical proteins provides a resource for functional discovery.

Giardia intestinalis is a globally prevalent cause of waterborne diarrheal disease, yet about 40% of its proteome remains functionally uncharacterized due to the lack of conserved homologous proteins and limited experimental validation of protein function. To begin addressing this gap, we created a large-scale subcellular localization resource by fluorescently tagging and imaging 608 Giardia proteins (12% of the proteome) expressed in live cells from native promoters. This dataset includes 240 hypothetical proteins, 215 domain-family proteins (including ankyrin repeat and NEK kinase families), 171 diplomonad- or Giardia-specific proteins, 69 conserved eukaryotic proteins, and 77 proteins with known functions that were previously unlocalized. Imaging revealed localization to cytoskeletal and Giardia-specific organelles (eight flagella, the ventral disk, and the median body), along with novel components of the plasma membrane and endomembrane systems. Integrating localization data with domain architecture, homology, and Giardia-specific Gene Ontology terms, we produced a "localization-informed" gene annotation with a standardized, structured nomenclature. This resource provides the largest experimentally validated functional annotation of the Giardia proteome to date, linking predicted gene models to cellular structures, creating testable hypotheses for protein function and establishing a durable framework for future studies of cell biology, pathogenesis, and eukaryotic evolution in this deeply divergent diplomonad lineage.