Bulk and selective autophagy cooperate to remodel a fungal proteome in response to changing nutrient availability

Cells remodel their proteomes in response to changing environments by coordinating protein synthesis and degradation. In yeast, degradation occurs via proteasomes and vacuoles, with bulk and selective autophagy supplying vacuolar cargo. Although these pathways are known, their relative contributions to proteome-wide remodeling remain unreported. To assess this, we developed a method (nPL-qMS) to pulse-label the methylotrophic yeast Komagataella phaffii (Pichia pastoris) with isotopically labeled nutrients that, when coupled to quantitative proteomics, enables global monitoring of protein degradation following an environmental perturbation. Genetic ablations revealed that autophagy drives most proteome remodeling upon nitrogen starvation, with minimal non-autophagic contributions. Cytosolic protein complexes, including ribosomes, are degraded through bulk autophagy, whereas degradation of peroxisomes and mitochondria uses selective autophagy. Notably, these pathways are independently regulated by environmental cues. Our approach expands known autophagic substrates, highlights autophagy's major role in fungal proteome remodeling, and provides rich resources and methods for future proteome remodeling studies.