Following chromosome segregation, the nuclear envelope (NE) must be reassembled and holes in the nuclear membrane must be "sealed." During NE assembly, the NE-specific adaptor, Cmp7, recruits/activates ESCRT-III proteins to mediate NE sealing. However, recent evidence suggests the presence of alternative mechanisms. In a screen using the fission yeast, S. japonicus, we recently implicated the ESCRT adaptor, Alx1, and a conserved, but little studied protein, Vid27, in Cmp7-independent NE assembly. Here, we provide direct evidence that Alx1 functions in a Cmp7- and ESCRT-independent NE assembly pathway via positive regulation of Vid27. Consistent with a role in membrane remodeling, Vid27 localizes to sites of postmitotic NE sealing and is essential in S. japonicus. Alx1 and Vid27 form a complex and mutations disrupting their interaction abolish Alx1's enhancement of Vid27 function at the NE. These findings define components of a new Cmp7- and ESCRT-independent NE assembly pathway, advancing our understanding of the mechanisms crucial for maintaining the integrity of the nucleus.

Lysosomal storage diseases (LSDs) comprise ~50 monogenic disorders marked by the buildup of cellular material in lysosomes, yet systematic global molecular phenotyping of proteins and lipids is lacking. We present a nanoflow-based multiomic single-shot technology (nMOST) workflow that quantifies HeLa cell proteomes and lipidomes from over two dozen LSD mutants. Global cross-correlation analysis between lipids and proteins identified autophagy defects, notably the accumulation of ferritinophagy substrates and receptors, especially in NPC1-/- and NPC2-/- mutants, where lysosomes accumulate cholesterol. Autophagic and endocytic cargo delivery failures correlated with elevated lysophosphatidylcholine species and multilamellar structures visualized by cryo-electron tomography. Loss of mitochondrial cristae, MICOS complex components, and OXPHOS components rich in iron-sulfur cluster proteins in NPC2-/- cells was largely alleviated when iron was provided through the transferrin system. This study reveals how lysosomal dysfunction affects mitochondrial homeostasis and underscores nMOST as a valuable discovery tool for identifying molecular phenotypes across LSDs.

The analysis of relative protein abundance has emerged as an important tool in cell biology. Typically, it is possible to quantify >8000 proteins under standard conditions. Tandem Mass Tags (TMT) are isobaric reagents that contain a set of isotopically distinct reporter ions, which can be used to quantify individual peptides in distinct samples through multiplexing(McAlister et al., 2014). Because the TMT analysis is performed in multiplexed format (up to 18 plex), it is possible to examine the effect of different perturbations (treatments, time courses, etc) on the total abundance of the proteome and include replicate samples as desired. This protocol is applicable to many different cell types, although the number of proteins quantified may differ, depending on the complexity of the proteomes in individual cell types. The small amount of protein needed (50-100 μg) makes application of this approach simple for many different types of cells. This protocol explicitly is used to examine the effects of nutrient stress on protein abundance in cell lines with or without autophagy.

During nutrient stress, macroautophagy degrades cellular macromolecules, thereby providing biosynthetic building blocks while simultaneously remodelling the proteome 1 , 2 . Although the machinery responsible for initiation of macroautophagy has been well characterized 3 , 4 , our understanding of the extent to which individual proteins, protein complexes and organelles are selected for autophagic degradation, and the underlying targeting mechanisms, is limited. Here we use orthogonal proteomic strategies to provide a spatial proteome census of autophagic cargo during nutrient stress in mammalian cells. We find that macroautophagy has selectivity for recycling membrane-bound organelles (principally Golgi and endoplasmic reticulum). Through autophagic cargo prioritization, we identify a complex of membrane-embedded proteins, YIPF3 and YIPF4, as receptors for Golgiphagy. During nutrient stress, YIPF3 and YIPF4 interact with ATG8 proteins through LIR motifs and are mobilized into autophagosomes that traffic to lysosomes in a process that requires the canonical autophagic machinery. Cells lacking YIPF3 or YIPF4 are selectively defective in elimination of a specific cohort of Golgi membrane proteins during nutrient stress. Moreover, YIPF3 and YIPF4 play an analogous role in Golgi remodelling during programmed conversion of stem cells to the neuronal lineage in vitro. Collectively, the findings of this study reveal prioritization of membrane protein cargo during nutrient-stress-dependent proteome remodelling and identify a Golgi remodelling pathway that requires membrane-embedded receptors.

Lysosomes are a major degradative organelle within eukaryotic cells. Previous work has developed a method wherein the TMEM192 protein is tagged on its C-terminus with an epitope tag in order to immunopurify (IP) lysosomes from cell extracts.1 This process is referred to as Lyso-IP. Such lysosomes can be used for proteomic analysis or for metabolomic analysis. The Lyso-IP is adapted from a previous reported method (Wyant et al., 2018). Here we also describe processing steps using proteomics after lysosome purification in the context of HeLa cells lacking the GRN gene. Such cells can be produced using the following protocol: DOI: dx.doi.org/10.17504/protocols.io.4r3l2oxqqv1y/v1.

ABSTRACT During nutrient stress, macroautophagy is employed to degrade cellular macromolecules, thereby providing biosynthetic building blocks while simultaneously remodeling the proteome. While the machinery responsible for initiation of macroautophagy is well characterized, our understanding of the extent to which individual proteins, protein complexes and organelles are selected for autophagic degradation, and the underlying targeting mechanisms is limited. Here, we use orthogonal proteomic strategies to provide a global molecular inventory of autophagic cargo during nutrient stress in mammalian cell lines. Through prioritization of autophagic cargo, we identify a heterodimeric pair of membrane-embedded proteins, YIPF3 and YIPF4, as receptors for Golgiphagy. During nutrient stress, YIPF4 is mobilized into ATG8-positive vesicles that traffic to lysosomes as measured via Golgiphagy flux reporters in a process that requires the VPS34 and ULK1-FIP200 arms of the autophagy system. Cells lacking YIPF3 or YIPF4 are selectively defective in elimination of Golgi membrane proteins during nutrient stress. By merging absolute protein abundance with autophagic turnover, we create a global protein census describing how autophagic degradation maps onto protein abundance and subcellular localization. Our results, available via an interactive web tool, reveal that autophagic turnover prioritizes membrane-bound organelles (principally Golgi and ER) for proteome remodeling during nutrient stress. One-Sentence Summary During nutrient stress, macroautophagy uses organelle-phagy receptors to prioritize recycling of Golgi and ER membrane proteins.

SUMMARY Degradation and recycling of plasma membrane-localized proteins occurs via the endolysosomal system, wherein endosomes bud into the cytosol from the plasma membrane and subsequently mature into degradative lysosomal compartments. The endolysosomal system also participates in intracellular membrane protein trafficking, signaling, and protein degradation via autophagy. Our understanding of the endolysosomal system has been significantly advanced through the development of methods for selective capture of lysosomes (termed Lyso-IP), but methods for analogous isolation of early intermediates in the endolysosomal system are lacking. Here, we develop an approach for rapid isolation of early/sorting endosomes through affinity capture of the early endosome-associated protein EEA1 (Endo-IP) and provide proteomic and lipidomic snapshots of early endosomes in action. In addition to resident endosomal components, we identify numerous recycling, regulatory and membrane fusion complexes as well as candidate cargo, providing a proteomic landscape of early/sorting endosomes that we distinguished from lysosomes via comparative proteomics. The approach allows capture of internalized transferrin within minutes of its addition to cells, indicating the utility of the approach for dynamic analysis of early/sorting endosomes. Among the endocytic cargo identified by Endo-IP was the amyloid precursor protein (APP) genetically linked with Alzheimer’s disease. Processing of APP to amyloidogenic Aβ peptides by β- and γ-Secretases can occur within the endolysosomal system among other compartments, but methods for spatial quantification of Aβ products in individual organelles are lacking. We combined Endo- and Lyso-IP with targeted proteomics to provide a spatial digital snapshot of Aβ products. This analysis revealed that products of Aβ processing by β- and γ-Secretases, and alterations in the specificity of cleavage by small molecule γ-Secretase modulators, can be quantified in both early/sorting endosomes and lysosomes. We anticipate that the Endo-IP approach will facilitate systematic interrogation of the many processes that are coordinated on early endosomes.

Lysosomes are subjected to physiological and pathophysiological insults over the course of their life cycle and are accordingly repaired or recycled. Lysophagy, the selective degradation of lysosomes via autophagy, occurs upon unrepairable lysosomal-membrane rupture; galectins bind to glycosylated macromolecules in the lysosome lumen, orchestrating a series of cellular responses to promote autophagic recycling of damaged lysosomes and transcriptional upregulation of lysosomal genes. Damaged lysosomes are ubiquitylated, resulting in the recruitment of ubiquitin-binding autophagy receptors, which promote assembly of an autophagosome around damaged lysosomes for delivery to healthy lysosomes for degradation. Here, we review the current state of our understanding of mechanisms used to mark and eliminate damaged lysosomes, and discuss the complexities of galectin function and ubiquitin-chain linkage types. Finally, we discuss the limitations of available data and challenges with the goal of understanding the mechanistic basis of key steps in lysophagic flux.

Degradation and recycling of plasma membrane proteins occurs via the endolysosomal system, wherein endosomes bud into the cytosol from the plasma membrane and subsequently mature into degradative lysosomal compartments. While methods have been developed for rapid selective capture of lysosomes (Lyso-IP), analogous methods for isolation of early endosome intermediates are lacking. Here, we develop an approach for rapid isolation of early/sorting endosomes through affinity capture of the early endosome-associated protein EEA1 (Endo-IP) and provide proteomic and lipidomic snapshots of EEA1-positive endosomes in action. We identify recycling, regulatory and membrane fusion complexes, as well as candidate cargo, providing a proteomic landscape of early/sorting endosomes. To demonstrate the utility of the method, we combined Endo- and Lyso-IP with multiplexed targeted proteomics to provide a spatial digital snapshot of amyloid precursor protein (APP) processing by β and γ-Secretases, which produce amyloidogenic Aβ species, and quantify small molecule modulation of Secretase action on endosomes. We anticipate that the Endo-IP approach will facilitate systematic interrogation of processes that are coordinated on EEA1-positive endosomes.

Haploinsufficiency of GRN causes frontotemporal dementia (FTD). The GRN locus produces progranulin (PGRN), which is cleaved to lysosomal granulin polypeptides. The function of lysosomal granulins and why their absence causes neurodegeneration are unclear. Here we discover that PGRN-deficient human cells and murine brains, as well as human frontal lobes from GRN-mutation FTD patients have increased levels of gangliosides, glycosphingolipids that contain sialic acid. In these cells and tissues, levels of lysosomal enzymes that catabolize gangliosides were normal, but levels of bis(monoacylglycero)phosphates (BMP), lipids required for ganglioside catabolism, were reduced with PGRN deficiency. Our findings indicate that granulins are required to maintain BMP levels to support ganglioside catabolism, and that PGRN deficiency in lysosomes leads to gangliosidosis. Lysosomal ganglioside accumulation may contribute to neuroinflammation and neurodegeneration susceptibility observed in FTD due to PGRN deficiency and other neurodegenerative diseases.

Lyso-IP is a method that allows for the isolation of lysosomes for proteomics and metabolomics using HA-tagged TMEM192 (dx.doi.org/10.17504/protocols.io.bybjpskn; dx.doi.org/10.17504/protocols.io.bx9hpr36). Here, we describe methods for cell line construction and maintenance of HeLa cells with TMEM192-3xHA with or without deletion of genes linked with lysosomal storage diseases.

SUMMARYHomozygous mutations of granulin precursor (GRN) lead to neuronal ceroid lipofuscinosis1, a severe neurodevelopmental disease, in humans and neuroinflammation in mice2. Haploinsufficiency of GRN almost invariably causes frontotemporal dementia (FTD)3,4. The GRN locus produces progranulin (PGRN), a lysosomal precursor protein that is cleaved to granulin peptides5,6. Despite intensive investigation, the function of granulins and the reason why their absence causes neurodegeneration remain unclear. Here, we investigated PGRN function in lipid degradation, a major function of lysosomes. We show that PGRN-knockout human cells, PGRN-deficient murine brain, and frontal lobes of human brains from patients with GRN mutation-related FTD have increased levels of gangliosides, highly abundant sialic acid–containing glycosphingolipids (GSL) that are degraded in lysosomes. Probing how PGRN deficiency causes these changes, we found normal levels and activities of enzymes that catabolize gangliosides. However, levels of bis(monoacylglycero)phosphate (BMP), a lysosomal lipid required for ganglioside catabolism7, were markedly reduced in PGRN-deficient cells and patient brain tissues. These data indicate that granulins are required to maintain BMP levels, which regulate ganglioside catabolism, and that PGRN deficiency in lysosomes leads to gangliosidosis. This aberrant accumulation of gangliosides may contribute to neuroinflammation and neurodegeneration susceptibility.

Lysosomes are a major degradative organelle within eukaryotic cells. Previous work has developed a method wherein the TMEM192 protein is tagged on its C-terminus with an epitope tag in order to immunopurify (IP) lysosomes from cell extracts.1 This process is referred to as Lyso-IP. Such lysosomes can be used for proteomic analysis or for metabolomic analysis. The Lyso-IP is adapted from a previous reported method (Wyant et al., 2018). Here we also describe processing steps using proteomics after lysosome purification in the context of lysosomal damaging agents. Agents such as L-Leucyl-L-Leucine methyl ester (hydrochloride) (LLoMe) and Gly-Phe-β-naphthylamide (GPN) induce lysosomal damage, leading to the degradation of damaged lysosomes by lysophagy. This adaptation of Lyso-IP provides a route to identify proteins that are recruited to damaged lysosomes using quantitative proteomics.

Removal of damaged organelles via the process of selective autophagy constitutes a major form of cellular quality control. Damaged organelles are recognized by a dedicated surveillance machinery, leading to the assembly of an autophagosome around the damaged organelle, prior to fusion with the degradative lysosomal compartment. Lysosomes themselves are also prone to damage and are degraded through the process of lysophagy. While early steps involve recognition of ruptured lysosomal membranes by glycan-binding galectins and ubiquitylation of transmembrane lysosomal proteins, many steps in the process, and their interrelationships, remain poorly understood, including the role and identity of cargo receptors required for completion of lysophagy. Here, we employ quantitative organelle capture and proximity biotinylation proteomics of autophagy adaptors, cargo receptors, and galectins in response to acute lysosomal damage, thereby revealing the landscape of lysosome-associated proteome remodeling during lysophagy. Among the proteins dynamically recruited to damaged lysosomes were ubiquitin-binding autophagic cargo receptors. Using newly developed lysophagic flux reporters including Lyso-Keima, we demonstrate that TAX1BP1, together with its associated kinase TBK1, are both necessary and sufficient to promote lysophagic flux in both HeLa cells and induced neurons (iNeurons). While the related receptor Optineurin (OPTN) can drive damage-dependent lysophagy when overexpressed, cells lacking either OPTN or CALCOCO2 still maintain significant lysophagic flux in HeLa cells. Mechanistically, TAX1BP1-driven lysophagy requires its N-terminal SKICH domain, which binds both TBK1 and the autophagy regulatory factor RB1CC1, and requires upstream ubiquitylation events for efficient recruitment and lysophagic flux. These results identify TAX1BP1 as a central component in the lysophagy pathway and provide a proteomic resource for future studies of the lysophagy process.

Lysophagy-the selective elimination of damaged lysosomes by the autophagy pathway-is a critical housekeeping mechanism in cells. This pathway surveils lysosomes and selectively demarcates terminally damaged lysosomes for elimination. Among the most upstream signaling proteins in this pathway are the glycan binding proteins-Galectins-which recognize N and O linked glycan chains on the luminal side of transmembrane lysosomal proteins. These glycosyl modifications are only accessible to galectin proteins upon extensive lysosomal membrane rupture and serve as a sensitive measure of lysosomal damage and eventual clearance by selective autophagy. Indeed, prior work has shown that immunofluorescence of Galectin-3 serves as a convenient proxy for lysophagic flux in tissue culture cells (Aits et al., 2015; Maejima et al., 2013). Here we describe a facile method for monitoring lysophagy using the acid sensitive fluorophore mKeima, affixed onto Galectin-3, which allows for the monitoring of lysophagic flux by Flow cytometry, Western blotting or Confocal imaging. This method, which we have termed Lyso-Keima, serves as a facile and quantitative assay for monitoring lysophagy in tissue culture cells.

Lysophagy-the selective elimination of damaged lysosomes by the autophagy pathway-is a critical housekeeping mechanism in cells. This pathway surveils lysosomes and selectively demarcates terminally damaged lysosomes for elimination. Among the most upstream signaling proteins in this pathway are the glycan binding proteins-Galectins-which recognize N and O linked glycan chains on the luminal side of transmembrane lysosomal proteins. These glycosyl modifications are only accessible to galectin proteins upon extensive lysosomal membrane rupture and serve as a sensitive measure of lysosomal damage and eventual clearance by selective autophagy. Indeed, prior work has shown that immunofluorescence of Galectin-3 serves as a convenient proxy for lysophagic flux in tissue culture cells (Aits et al., 2015; Maejima et al., 2013). Here we describe a method for monitoring protein recruitment to damaged lysosomes via immunofluorescence and confocal imaging.

Lysophagy-the selective elimination of damaged lysosomes by the autophagy pathway-is a critical housekeeping mechanism in cells. This pathway surveils lysosomes and selectively demarcates terminally damaged lysosomes for elimination. Among the most upstream signaling proteins in this pathway are the glycan binding proteins-Galectins-which recognize N and O linked glycan chains on the luminal side of transmembrane lysosomal proteins. These glycosyl modifications are only accessible to galectin proteins upon extensive lysosomal membrane rupture and serve as a sensitive measure of lysosomal damage and eventual clearance by selective autophagy. Indeed, prior work has shown that immunofluorescence of Galectin-3 serves as a convenient proxy for lysophagic flux in tissue culture cells (Aits et al., 2015; Maejima et al., 2013). Here we describe our method for monitoring galectin-3 puncta clearance as a proxy for turnover of damaged lysosomes via immunofluorescence and confocal imaging.

Lysosomes are a major degradative organelle within eukaryotic cells. Previous work has developed a method wherein the TMEM192 protein is tagged on its C-terminus with an epitope tag in order to immunopurify (IP) lysosomes from cell extracts.1 This process is referred to as Lyso-IP. Such lysosomes can be used for proteomic analysis or for metabolomic analysis. A detailed protocol has been described by Dong et al (2021)2 for the isolation of lysosomes (https://protocols.io/view/sample-preparation-protocol-for-lipidomics-harvest-br9ym97w), with an emphasis on downstream analysis by metabolomics. Here we describe processing steps using proteomics after lysosome purification in the context of lysosomal damaging agents. Agents such as L-Leucyl-L-Leucine methyl ester (hydrochloride) (LLoMe) and Gly-Phe-β-naphthylamide (GPN) induce lysosomal damage, leading to the degradation of damaged lysosomes by lysophagy. This adaptation of Lyso-IP provides a route to identify proteins that are recruited to damaged lysosomes using quantitative proteomics.

Numerous mutations that impair retrograde membrane trafficking between endosomes and the Golgi apparatus lead to neurodegenerative diseases. For example, mutations in the endosomal retromer complex are implicated in Alzheimer’s and Parkinson’s diseases, and mutations of the Golgi-associated retrograde protein (GARP) complex cause progressive cerebello-cerebral atrophy type 2 (PCCA2). However, how these mutations cause neurodegeneration is unknown. GARP mutations in yeast, including one causing PCCA2, result in sphingolipid abnormalities and impaired cell growth that are corrected by treatment with myriocin, a sphingolipid synthesis inhibitor, suggesting that alterations in sphingolipid metabolism contribute to cell dysfunction and death. Here we tested this hypothesis in wobbler mice, a murine model with a homozygous partial loss-of-function mutation in Vps54 (GARP protein) that causes motor neuron disease. Cytotoxic sphingoid long-chain bases accumulated in embryonic fibroblasts and spinal cords from wobbler mice. Remarkably, chronic treatment of wobbler mice with myriocin markedly improved their wellness scores, grip strength, neuropathology, and survival. Proteomic analyses of wobbler fibroblasts revealed extensive missorting of lysosomal proteins, including sphingolipid catabolism enzymes, to the Golgi compartment, which may contribute to the sphingolipid abnormalities. Our findings establish that altered sphingolipid metabolism due to GARP mutations contributes to neurodegeneration and suggest that inhibiting sphingolipid synthesis might provide a useful strategy for treating these disorders.

Affinity purification–mass spectrometry elucidates protein interaction networks and co-complexes to build, to our knowledge, the largest experimentally derived human protein interaction network so far, termed BioPlex 2.0. The thousands of proteins within a cell function as modules and networks to coordinate their biological activities. Large-scale efforts are underway to build protein interaction maps that reveal cellular proteome architecture. Here, Wade Harper and colleagues use affinity purification mass spectrometry to elucidate protein interaction networks and co-complexes and build the largest experimentally derived human proteome interaction network to date, termed BioPlex 2.0. Containing over 29,000 novel co-associations and 1,300 protein communities representing diverse cellular activities, BioPlex 2.0 is more than double the size of their earlier interaction network BioPlex 1.0 and will be a valuable resource for exploring uncharacterized proteins and candidate disease-linked genes. The physiology of a cell can be viewed as the product of thousands of proteins acting in concert to shape the cellular response. Coordination is achieved in part through networks of protein–protein interactions that assemble functionally related proteins into complexes, organelles, and signal transduction pathways. Understanding the architecture of the human proteome has the potential to inform cellular, structural, and evolutionary mechanisms and is critical to elucidating how genome variation contributes to disease1,2,3. Here we present BioPlex 2.0 (Biophysical Interactions of ORFeome-derived complexes), which uses robust affinity purification–mass spectrometry methodology4 to elucidate protein interaction networks and co-complexes nucleated by more than 25% of protein-coding genes from the human genome, and constitutes, to our knowledge, the largest such network so far. With more than 56,000 candidate interactions, BioPlex 2.0 contains more than 29,000 previously unknown co-associations and provides functional insights into hundreds of poorly characterized proteins while enhancing network-based analyses of domain associations, subcellular localization, and co-complex formation. Unsupervised Markov clustering5 of interacting proteins identified more than 1,300 protein communities representing diverse cellular activities. Genes essential for cell fitness6,7 are enriched within 53 communities representing central cellular functions. Moreover, we identified 442 communities associated with more than 2,000 disease annotations, placing numerous candidate disease genes into a cellular framework. BioPlex 2.0 exceeds previous experimentally derived interaction networks in depth and breadth, and will be a valuable resource for exploring the biology of incompletely characterized proteins and for elucidating larger-scale patterns of proteome organization.