ER-phagy, the selective autophagy of endoplasmic reticulum (ER), safeguards organelle homeostasis by eliminating misfolded proteins and regulating ER size. ER-phagy can occur by macroautophagic and microautophagic mechanisms. While dedicated machinery for macro-ER-phagy has been discovered, the molecules and mechanisms mediating micro-ER-phagy remain unknown. Here, we first show that micro-ER-phagy in yeast involves the conversion of stacked cisternal ER into multilamellar ER whorls during microautophagic uptake into lysosomes. Second, we identify the conserved Nem1-Spo7 phosphatase complex and the ESCRT machinery as key components for micro-ER-phagy. Third, we demonstrate that macro-and micro-ER-phagy are parallel pathways with distinct molecular requirements. Finally, we provide evidence that the ESCRT machinery directly functions in scission of the lysosomal membrane to complete the microautophagic uptake of ER. These findings establish a framework for a mechanistic understanding of micro-ER-phagy and, thus, a comprehensive appreciation of the role of autophagy in ER homeostasis.

Summary Most mitochondrial proteins are translated in the cytosol and imported into mitochondria. Mutations in the mitochondrial protein import machinery cause human pathologies. However, a lack of suitable tools to measure protein uptake across the mitochondrial proteome has prevented the identification of specific proteins affected by import perturbation. Here, we introduce mePROD mt , a pulsed-SILAC based proteomics approach that includes a booster signal to increase the sensitivity for mitochondrial proteins selectively, enabling global dynamic analysis of endogenous mitochondrial protein uptake in cells. We applied mePROD mt to determine protein uptake kinetics and examined how inhibitors of mitochondrial import machineries affect protein uptake. Monitoring changes in translation and uptake upon mitochondrial membrane depolarization revealed that protein uptake was extensively modulated by the import and translation machineries via activation of the integrated stress response. Strikingly, uptake changes were not uniform, with subsets of proteins being unaffected or decreased due to changes in translation or import capacity.

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Effective therapy of wounds is difficult, especially for chronic, non-healing wounds, and novel therapeutics are urgently needed. This challenge can be addressed with bioactive wound dressings providing a microenvironment and facilitating cell proliferation and migration, ideally incorporating actives, which initiate and/or progress effective healing upon release. In this context, electrospun scaffolds loaded with growth factors emerged as promising wound dressings due to their biocompatibility, similarity to the extracellular matrix, and potential for controlled drug release. In this study, electrospun core–shell fibers were designed composed of a combination of polycaprolactone and polyethylene oxide. Insulin, a proteohormone with growth factor characteristics, was successfully incorporated into the core and was released in a controlled manner. The fibers exhibited favorable mechanical properties and a surface guiding cell migration for wound closure in combination with a high uptake capacity for wound exudate. Biocompatibility and significant wound healing effects were shown in interaction studies with human skin cells. As a new approach, analysis of the wound proteome in treated ex vivo human skin wounds clearly demonstrated a remarkable increase in wound healing biomarkers. Based on these findings, insulin-loaded electrospun wound dressings bear a high potential as effective wound healing therapeutics overcoming current challenges in the clinics.