Membrane dynamics and protein targets of lipid droplet microautophagy during ER stress-induced proteostasis in the budding yeast, Saccharomyces cerevisiae

ABSTRACT

Our previous studies reveal a mechanism for lipid droplet (LD)-mediated proteostasis in the endoplasmic reticulum (ER) whereby unfolded proteins that accumulate in the ER in response to lipid imbalance-induced ER stress are removed by LDs and degraded by microlipophagy (µLP), autophagosome-independent LD uptake into the vacuole (the yeast lysosome). Here, we show that dithiothreitol- or tunicamycin-induced ER stress also induces µLP and identify an unexpected role for vacuolar membrane dynamics in this process. All stressors studied induce vacuolar fragmentation prior to µLP. Moreover, during µLP, fragmented vacuoles fuse to form cup-shaped structures that encapsulate and ultimately take up LDs. Our studies also indicate that proteins of the endosome sorting complexes required for transport (ESCRT) are upregulated, required for µLP, and recruited to LDs, vacuolar membranes, and sites of vacuolar membrane scission during µLP. We identify possible target proteins for LD-mediated ER proteostasis. Our live-cell imaging studies reveal that one potential target (Nup159) localizes to punctate structures that colocalizes with LDs 1) during movement from ER membranes to the cytosol, 2) during microautophagic uptake into vacuoles, and 3) within the vacuolar lumen. Finally, we find that mutations that inhibit LD biogenesis, homotypic vacuolar membrane fusion or ESCRT function inhibit stress-induced autophagy of Nup159 and other ER proteins. Thus, we have obtained the first direct evidence that LDs and µLP can mediate ER stress-induced ER proteostasis, and identified direct roles for ESCRT and vacuolar membrane fusion in that process.

KEYWORDS:

• ER stress
• erad
• escrt
• lipid droplet proteome
• microautophagy
• microlipophagy
• unfolded protein response
• vacuolar membrane fusion
• vacuole

Abbreviations:

AAA-ATPase: ATPase associated with diverse cellular activities; ATG: autophagy-related; CMAC: 7-amino-4-chloromethylcoumarin; DTT: dithiothreitol; ER: endoplasmic reticulum; ERAD: ER-associated degradation; eroGFP: ER-targeted, redox-sensing GFP; ESCRT: endosome sorting complexes required for transport; GFPEnvy: green fluorescent protein Envy; LD: lipid droplet; µLP: microlipophagy; MVB: multivesicular body; nER: nuclear ER; PC: phosphatidylcholine; PE: phosphatidylethanolamine; RT: room temperature; SC: synthetic complete; sfGFP: superfolder GFP; TG: triglycerides; TM: tunicamycin; UPR: unfolded protein response; YPD: yeast-peptone-dextrose.

Acknowledgments

We thank the members of the Pon laboratory as well as Drs. Ai Yamamoto, Howard Worman, Eric Schon, Theresa Swayne and Rajesh Soni (Columbia University) for support and valuable discussion. We also thank Dr. William Wickner (Dartmouth College) for support, invaluable advice and yeast strains for the work on homotypic vacuolar fusion. This work was supported by awards from the National Institutes of Health (NIH) (GM122589 and AG051047) and Muscular Dystrophy Association (MDA 314107) to LAP, NIH 5T32 GM007367 and 1F31 AR070013 to EJG, Howard Hughes Medical Institute (HHMI 56006760) to JDV and NIH/NCRR 1S10RR023454 to JMM. We thank the Proteomics Shared Resource for mass spectrometry, and the Confocal and Specialized Microscopy Shared Resource for assistance with time-lapse microscopy and image analysis. Both cores are in the Herbert Irving Comprehensive Cancer Center at Columbia University Medical Center, which is supported in part by an award from the NIH/NCI (5 P30 CA13696).

Data availability statement

The data that support the findings of this study are available from the corresponding author, L.P., upon reasonable request.

Disclosure statement

The authors declare no competing interests.

Supplementary material

Supplemental data for this article can be accessed here.

Additional information

Funding

This work was supported by the Foundation for the National Institutes of Health [GM45735]; Foundation for the National Institutes of Health [GM122589]; Foundation for the National Institutes of Health [AG051047]; Foundation for the National Institutes of Health [GM007367]; Foundation for the National Institutes of Health [AG055326]; Foundation for the National Institutes of Health [1S10RR023454]; Foundation for the National Institutes of Health [AR070013]; Howard Hughes Medical Institute [HHMI 56006760]; Muscular Dystrophy Association [MDA 314107].