Decoding the cold: integrated secretome profiling reveals extracellular adaptation mechanisms in Glaciozyma antarctica.

Glaciozyma antarctica is an Antarctic psychrophilic yeast providing an excellent eukaryotic model for dissecting molecular mechanisms of cold adaptation. Yet, its extracellular protein repertoire, the interface between the cell and extreme surroundings, remains unexplored. Here, we present the first integrated bioinformatics-proteomics secretome map of G. antarctica, linking computational prediction with experimental validation under varying temperature stress, yielding 9 high-confidence experimentally supported secreted proteins. Three complementary pipelines (Phobius, SignalP/TMHMM, and WoLF PSORT) predicted 278 high-confidence secreted proteins, predominantly glycosyl hydrolases, lipases, oxidoreductases, and stress-response factors. To validate these predictions, we performed LC-MS/MS analysis of extracellular fractions from cultures exposed to - 12 °C, 0 °C, and 12 °C. A total of 104 secreted proteins was identified, including 55 shared across all temperatures and multiple unique cold-induced candidates such as antifreeze proteins and glycosidases. Comparative analysis uncovered a temperature-dependent remodeling of the secretome and revealed several proteins lacking canonical signal peptides, highlighting potential unconventional secretion mechanisms or moonlighting roles in extracellular environments. These findings demonstrate that G. antarctica adapts to freezing environments by dynamically reshaping its extracellular proteome to maintain structural plasticity and metabolic efficiency. This integrative approach identifies promising cold-active enzymes for potential application in the low-temperature biocatalysis industry.