Nuclear Microautophagy Drives Vacuolar Targeting of Yeast Iron-Regulated Proteins During Lipid and Iron Limitation

Iron is an essential cofactor involved in cellular processes, including energy generation and the biosynthesis of DNA, proteins, and lipids. The limited solubility of iron at physiological pH frequently results in iron deficiency, thus necessitating sophisticated regulatory mechanisms to maintain iron homeostasis. In Saccharomyces cerevisiae, the transcription factor Aft1 mediates the early response to iron limitation by accumulating in the nucleus and activating the iron regulon, a set of genes involved in iron uptake, utilization and sparing. One of Aft1 targets, CTH2, encodes for a protein that promotes iron economy by post-transcriptionally downregulating non-essential iron-dependent pathways. Yeast cells that exhibit defects in unsaturated fatty acid (UFA) biosynthesis, such as mga2Δ mutants, mislocalize Aft1 to the vacuole under iron-deficient conditions, which impairs activation of the iron regulon. In this study, we show that Cth2, but not other nucleo-cytoplasmic shuttling proteins, also accumulates in the vacuole under simultaneous UFA and iron deficiencies. The deletion of autophagy- and piecemeal microautophagy of the nucleus (PMN)-related genes, including ATG1 and NVJ1, prevents Aft1 vacuolar mislocalization. Furthermore, the subcellular distribution of Nvj1 supports PMN activation under these conditions. Despite preventing vacuolar accumulation, these mutations do not restore the regulatory functions of Aft1 and Cth2, nor do they rescue growth in low-iron conditions. These findings suggest that PMN selectively targets non-functional iron-regulated proteins for degradation when both iron and UFA levels are limiting, serving as a quality control mechanism rather than a pathway for functional recovery. These findings underscore a regulatory layer coordinating nutrient sensing and protein turnover.