Lysosomes rely on their resident transporter proteins to return products of catabolism to the cell for reuse and for cellular signaling, metal storage, and maintaining the lumenal environment. Despite their importance, little is known about the lifetime of these transporters or how they are regulated. Using Saccharomyces cerevisiae as a model, we discovered a new pathway intrinsic to homotypic lysosome membrane fusion that is responsible for their degradation. Transporter proteins are selectively sorted by the docking machinery into an area between apposing lysosome membranes, which is internalized and degraded by lumenal hydrolases upon organelle fusion. These proteins have diverse lifetimes that are regulated in response to protein misfolding, changing substrate levels, or TOR activation. Analogous to endocytosis for controlling surface protein levels, the "intralumenal fragment pathway" is critical for lysosome membrane remodeling required for organelle function in the context of cellular protein quality control, ion homeostasis, and metabolism.

When vacuolar lysosomes fuse, an intralumenal membrane fragment is produced and degraded by hydrolyses. How or why this fragment forms is not entirely understood. We show that the fusion machinery regulates stalk expansion during lipid bilayer fusion to create or eliminate fragments, affecting lysosome morphology and transporter protein turnover.

Surface receptor and transporter protein down-regulation is assumed to be exclusively mediated by the canonical multivesicular body (MVB) pathway and ESCRTs (Endosomal Sorting Complexes Required for Transport). However, few surface proteins are known to require ESCRTs for down-regulation, and reports of ESCRT-independent degradation are emerging, suggesting that alternative pathways exist. Here, using Saccharomyces cerevisiae as a model, we show that the hexose transporter Hxt3 does not require ESCRTs for down-regulation conferring resistance to 2-deoxyglucose. This is consistent with GFP-tagged Hxt3 bypassing ESCRT-mediated entry into intralumenal vesicles at endosomes. Instead, Hxt3-GFP accumulates on vacuolar lysosome membranes and is sorted into an area that, upon fusion, is internalized as an intralumenal fragment (ILF) and degraded. Moreover, heat stress or cycloheximide trigger degradation of Hxt3-GFP and other surface transporter proteins (Itr1, Aqr1) by this ESCRT-independent process. How this ILF pathway compares to the MVB pathway and potentially contributes to physiology is discussed.

Upon vacuolar lysosome (or vacuole) fusion in S. cerevisiae, a portion of membrane is internalized and catabolized. Formation of this intralumenal fragment (ILF) is important for organelle protein and lipid homeostasis and remodeling. But how ILF formation is optimized for membrane turnover is not understood. Here, we show that fewer ILFs form when the interaction between the Rab-GTPase Ypt7 and its effector Vps41 (a subunit of the tethering complex HOPS) is interrupted by a point mutation (Ypt7-D44N). Subsequent phosphorylation of Vps41 by the casein kinase Yck3 prevents stabilization of trans-SNARE complexes needed for lipid bilayer pore formation. Impairing ILF formation prevents clearance of misfolded proteins from vacuole membranes and promotes organelle permeability and cell death. We propose that HOPS coordinates Rab, kinase, and SNARE cycles to modulate ILF size during vacuole fusion, regulating lipid and protein turnover important for quality control and membrane integrity.

Diverse physiology relies on receptor and transporter protein down–regulation and degradation mediated by ESCRTs. Loss–of–function mutations in human ESCRT genes linked to cancers and neurological disorders are thought to block this process. However, when homologous mutations are introduced into model organisms, cells thrive and degradation persists, suggesting other mechanisms compensate. To better understand this secondary process, we studied degradation of transporter (Mup1) or receptor (Ste3) proteins when ESCRT genes (VPS27, VPS36) are deleted in Saccharomyces cerevisiae using live-cell imaging and organelle biochemistry. We find that endocytosis remains intact, but internalized proteins aberrantly accumulate on vacuolar lysosome membranes within cells. Here they are sorted for degradation by the intralumenal fragment (ILF) pathway, constitutively or when triggered by substrates, misfolding or TOR activation in vivo and in vitro. Thus, the ILF pathway functions as fail–safe layer of defense when ESCRTs disregard their clients, representing a two–tiered system that ensures degradation of surface polytopic proteins.