Organelle Proteomics Research Articles

Precision proteomics with TurboID: mapping the suborganelle landscape.

Recent research underscores the pivotal role of cellular organelles, such as mitochondria, the endoplasmic reticulum, and lysosomes, in maintaining cellular homeostasis. Their dynamic interactions are critical for metabolic regulation and stress response. Analysis of organelle proteomes offers valuable insights into their functions in both physiology and disease. Traditional proteomic approaches to studying isolated organelles are now complemented by innovative methodologies focusing on inter-organelle interactions. This review examines the integration of advanced proximity labeling technologies, including TurboID and split-TurboID, which address the inherent limitations of traditional techniques and enable precision proteomics of suborganelle compartments and inter-organellar contact sites. These innovations have led to discoveries regarding organelle interconnections, revealing mechanisms underlying metabolic processes such as cholesterol metabolism, glucose metabolism, and lysosomal repair. In addition to highlighting the advancements in TurboID applications, this review delineates the evolving trends in organelle research, underscoring the transformative potential of these techniques to significantly enhance organelle-specific proteomic investigations.

Evolutionary trajectory for nuclear functions of ciliary transport complex proteins.

SUMMARYCilia and the nucleus were two defining features of the last eukaryotic common ancestor. In early eukaryotic evolution, these structures evolved through the diversification of a common membrane-coating ancestor, the protocoatomer. While in cilia, the descendants of this protein complex evolved into parts of the intraflagellar transport complexes and BBSome, the nucleus gained its selectivity by recruiting protocoatomer-like proteins to the nuclear envelope to form the selective nuclear pore complexes. Recent studies show a growing number of proteins shared between the proteomes of the respective organelles, and it is currently unknown how ciliary transport proteins could acquire nuclear functions and vice versa. The nuclear functions of ciliary proteins are still observable today and remain relevant for the understanding of the disease mechanisms behind ciliopathies. In this work, we review the evolutionary history of cilia and nucleus and their respective defining proteins and integrate current knowledge into theories for early eukaryotic evolution. We postulate a scenario where both compartments co-evolved and that fits current models of eukaryotic evolution, explaining how ciliary proteins and nucleoporins acquired their dual functions.

Molecular Chaperonin HSP60: Current Understanding and Future Prospects.

Molecular chaperones are highly conserved across evolution and play a crucial role in preserving protein homeostasis. The 60 kDa heat shock protein (HSP60), also referred to as chaperonin 60 (Cpn60), resides within mitochondria and is involved in maintaining the organelle's proteome integrity and homeostasis. The HSP60 family, encompassing Cpn60, plays diverse roles in cellular processes, including protein folding, cell signaling, and managing high-temperature stress. In prokaryotes, HSP60 is well understood as a GroEL/GroES complex, which forms a double-ring cavity and aids in protein folding. In eukaryotes, HSP60 is implicated in numerous biological functions, like facilitating the folding of native proteins and influencing disease and development processes. Notably, research highlights its critical involvement in sustaining oxidative stress and preserving mitochondrial integrity. HSP60 perturbation results in the loss of the mitochondria integrity and activates apoptosis. Currently, numerous clinical investigations are in progress to explore targeting HSP60 both in vivo and in vitro across various disease models. These studies aim to enhance our comprehension of disease mechanisms and potentially harness HSP60 as a therapeutic target for various conditions, including cancer, inflammatory disorders, and neurodegenerative diseases. This review delves into the diverse functions of HSP60 in regulating proteo-homeostasis, oxidative stress, ROS, apoptosis, and its implications in diseases like cancer and neurodegeneration.

A molecular atlas of plastid and mitochondrial proteins reveals organellar remodeling during plant evolutionary transitions from algae to angiosperms.

Algae and plants carry 2 organelles of endosymbiotic origin that have been co-evolving in their host cells for more than a billion years. The biology of plastids and mitochondria can differ significantly across major lineages and organelle changes likely accompanied the adaptation to new ecological niches such as the terrestrial habitat. Based on organelle proteome data and the genomes of 168 phototrophic (Archaeplastida) versus a broad range of 518 non-phototrophic eukaryotes, we screened for changes in plastid and mitochondrial biology across 1 billion years of evolution. Taking into account 331,571 protein families (or orthogroups), we identify 31,625 protein families that are unique to primary plastid-bearing eukaryotes. The 1,906 and 825 protein families are predicted to operate in plastids and mitochondria, respectively. Tracing the evolutionary history of these protein families through evolutionary time uncovers the significant remodeling the organelles experienced from algae to land plants. The analyses of gained orthogroups identifies molecular changes of organelle biology that connect to the diversification of major lineages and facilitated major transitions from chlorophytes en route to the global greening and origin of angiosperms.

The ion channels of endomembranes.

The endomembrane system consists of organellar membranes in the biosynthetic pathway [endoplasmic reticulum (ER), Golgi apparatus, and secretory vesicles] as well as those in the degradative pathway (early endosomes, macropinosomes, phagosomes, autophagosomes, late endosomes, and lysosomes). These endomembrane organelles/vesicles work together to synthesize, modify, package, transport, and degrade proteins, carbohydrates, and lipids, regulating the balance between cellular anabolism and catabolism. Large ion concentration gradients exist across endomembranes: Ca2+ gradients for most endomembrane organelles and H+ gradients for the acidic compartments. Ion (Na+, K+, H+, Ca2+, and Cl-) channels on the organellar membranes control ion flux in response to cellular cues, allowing rapid informational exchange between the cytosol and organelle lumen. Recent advances in organelle proteomics, organellar electrophysiology, and luminal and juxtaorganellar ion imaging have led to molecular identification and functional characterization of about two dozen endomembrane ion channels. For example, whereas IP3R1-3 channels mediate Ca2+ release from the ER in response to neurotransmitter and hormone stimulation, TRPML1-3 and TMEM175 channels mediate lysosomal Ca2+ and H+ release, respectively, in response to nutritional and trafficking cues. This review aims to summarize the current understanding of these endomembrane channels, with a focus on their subcellular localizations, ion permeation properties, gating mechanisms, cell biological functions, and disease relevance.

Monitoring the in vitro import and assembly of mitochondrial precursor proteins into mammalian mitochondria.

Mitochondrial protein import is a complex process governing the delivery of the organelle's proteome. This process, in turn, is essential for maintaining mitochondrial function and cellular homeostasis. Initiated by protein synthesis in the cytoplasm, precursor proteins destined for the mitochondria possess targeting signals that guide them to the mitochondrial surface. At mitochondria, the translocation of proteins across the mitochondrial membranes involves an intricate interplay between translocases, chaperones, and receptors. The mitochondrial import assay offers researchers the opportunity to recapitulate the process of protein import in vitro. The assay has served as an indispensable tool in helping decipher the intricacies of protein translocation into mitochondria, first in fungal models, and subsequently in higher eukaryotic models. In this chapter, we will describe how protein import can be assayed using mammalian mitochondria and provide insight into the types of questions that can be addressed in mammalian mitochondrial biology using this experimental approach.

Improved methods for total and chloroplast protein extraction from Cajanus species for two-dimensional gel electrophoresis and mass spectrometry.

The recent advances in pigeon pea genomics, including high-quality whole genome and chloroplast genome sequence information helped develop improved varieties. However, a comprehensive Cajanus proteome, including the organelle proteome, is yet to be fully mapped. The spatial delineation of pigeon pea proteins at sub-cellular levels and inter-organelle communication could offer valuable insights into its defense mechanism against various stresses. However, the major bottleneck in the proteomic study is the lack of a suitable method of protein extraction and sample preparation compatible with two-dimensional gel electrophoresis (2D-PAGE), liquid chromatography-mass spectrometry (LCMS), or matrix-assisted laser desorption ionization-time of flight (MALDi-ToF). Our study introduces two efficient methods, one for isolating total proteins and another for organelle (chloroplast) proteins from various Cajanus spp. For total protein extraction, we have optimized a protocol using phenol in combination with a reducing agent (DTT) and protease inhibitor cocktail, also washing (6-7 times) with ice-cold acetone after overnight protein precipitation of total proteins. Our modified extraction method using phenol for total leaf protein yielded approximately 2-fold more proteins than the previously reported protocols from C. cajan (3.18 ± 0.11 mg/gm) and C. scarabaeoides (2.06 ± 0.08 mg/gm). We have also optimized a protocol for plastid protein extraction, which yielded 1.33 ± 0.25 mg/10 gm plastid proteins from C. cajan and 0.88 ± 0.19 mg/10 gm plastid proteins from C. scarabaeoides. The 2D-PAGE analysis revealed 678 ± 08 reproducible total protein spots from C. cajan and 597 ± 22 protein spots from C. scarabaeoides. Similarly, we found 566 ± 10 and 486 ± 14 reproducible chloroplast protein spots in C. cajan and C. scarabaeoides, respectively. We confirmed the plastid protein fractions through immunoblot analysis using antibodies against LHCb1/LHCⅡ type Ⅰ protein. We found both methods suitable for 2D-PAGE and mass spectrometry (MS). This is the first report on developing protocols for total and chloroplastic protein extraction of Cajanus spp. suitable for advanced proteomics research.

Proteomic approach to investigate the expression, localization, and functions of the protein product of the <i>SOWAHD</i> gene during granulocytic differentiation

Cataloging human proteins, determining their level of content, cellular localization, function performed, and potential medical significance are important tasks facing the global proteomic community. At present, the localization and functions of protein products for almost half of the protein-coding genes are unknown or poorly understood. The study of the organelle proteome is a promising approach to reveal the localization and functions of human proteins. The nuclear proteome is of particular interest because many nuclear-localized proteins, such as transcription factors, perform regulatory functions that determine cell fate. According to the results of a meta-analysis of the nuclear proteome, or nucleome, of HL-60 cells treated by all-trans-retinoic acid (ATRA), it was revealed that the function and localization of the protein product of the SOWAHD gene is poorly understood, in addition, there is no comprehensive information on the expression of SOWAHD at the protein level. In HL-60 cells for the protein-coding gene SOWAHD, mRNA expression was determined at the level of 6.4 ± 0.7 transcripts per million molecules. Using targeted mass spectrometry, the content of SOWAHD protein was measured in the range of 0.27-1.25 fmol/µg of total protein. Using stable isotope pulse-chase labeling, the half-life for the protein product of the SOWAHD gene was determined to be approximately 19 h. Proteomic profiling of the nuclear fraction of HL-60 cells showed that the SOWAHD protein content increased during ATRA-induced granulocytic differentiation, peaking at 9 h after the addition of the inductor and followed by a decrease at later time points. The results of the study indicate for the first time the nuclear localization and involvement of the protein product of the SOWAHD gene in induced granulocytic differentiation.

Proteomic Approach to Investigating Expression, Localization, and Functions of the SOWAHD Gene Protein Product during Granulocytic Differentiation.

Cataloging human proteins and evaluation of their expression, cellular localization, functions, and potential medical significance are important tasks for the global proteomic community. At present, localization and functions of protein products for almost half of protein-coding genes remain unknown or poorly understood. Investigation of organelle proteomes is a promising approach to uncovering localization and functions of human proteins. Nuclear proteome is of particular interest because many nuclear proteins, e.g., transcription factors, regulate functions that determine cell fate. Meta-analysis of the nuclear proteome, or nucleome, of HL-60 cells treated with all-trans-retinoic acid (ATRA) has shown that the functions and localization of a protein product of the SOWAHD gene are poorly understood. Also, there is no comprehensive information on the SOWAHD gene expression at the protein level. In HL-60 cells, the number of mRNA transcripts of the SOWAHD gene was determined as 6.4 ± 0.7 transcripts per million molecules. Using targeted mass spectrometry, the content of the SOWAHD protein was measured as 0.27 to 1.25 fmol/μg total protein. The half-life for the protein product of the SOWAHD gene determined using stable isotope pulse-chase labeling was ~19 h. Proteomic profiling of the nuclear fraction of HL-60 cells showed that the content of the SOWAHD protein increased during the ATRA-induced granulocytic differentiation, reached the peak value at 9 h after ATRA addition, and then decreased. Nuclear location and involvement of the SOWAHD protein in the ATRA-induced granulocytic differentiation have been demonstrated for the first time.

Expression of malic enzyme reveals subcellular carbon partitioning for storage reserve production in soybeans.

Central metabolism produces amino and fatty acids for protein and lipids that establish seed value. Biosynthesis of storage reserves occurs in multiple organelles that exchange central intermediates including two essential metabolites, malate, and pyruvate that are linked by malic enzyme. Malic enzyme can be active in multiple subcellular compartments, partitioning carbon and reducing equivalents for anabolic and catabolic requirements. Prior studies based on isotopic labeling and steady-state metabolic flux analyses indicated malic enzyme provides carbon for fatty acid biosynthesis in plants, though genetic evidence confirming this role is lacking. We hypothesized that increasing malic enzyme flux would alter carbon partitioning and result in increased lipid levels in soybeans. Homozygous transgenic soybean plants expressing Arabidopsis malic enzyme alleles, targeting the translational products to plastid or outside the plastid during seed development, were verified by transcript and enzyme activity analyses, organelle proteomics, and transient expression assays. Protein, oil, central metabolites, cofactors, and acyl-acyl carrier protein (ACPs) levels were quantified overdevelopment. Amino and fatty acid levels were altered resulting in an increase in lipids by 0.5-2% of seed biomass (i.e. 2-9% change in oil). Subcellular targeting of a single gene product in central metabolism impacts carbon and reducing equivalent partitioning for seed storage reserves in soybeans.

A proteomic analysis shows the stimulation of light reactions and inhibition of the Calvin cycle in the skin chloroplasts of ripe red grape berries.

The role of photosynthesis in fruits still challenges scientists. This is especially true in the case of mature grape berries of red varieties lined by an anthocyanin-enriched exocarp (skin) almost impermeable to gases. Although chlorophylls are degraded and replaced by carotenoids in several fruits, available evidence suggests that they may persist in red grapes at maturity. In the present study, chloroplasts were isolated from the skin of red grape berries (cv. Vinhão) to measure chlorophyll levels and the organelle proteome. The results showed that chloroplasts (and chlorophylls) are maintained in ripe berries masked by anthocyanin accumulation and that the proteome of chloroplasts from green and mature berries is distinct. Several proteins of the light reactions significantly accumulated in chloroplasts at the mature stage including those of light-harvesting complexes of photosystems I (PSI) and II (PSII), redox chain, and ATP synthase, while chloroplasts at the green stage accumulated more proteins involved in the Calvin cycle and the biosynthesis of amino acids, including precursors of secondary metabolism. Taken together, results suggest that although chloroplasts are more involved in biosynthetic reactions in green berries, at the mature stage, they may provide ATP for cell maintenance and metabolism or even O2 to feed the respiratory demand of inner tissues.

Vitamin C modulates the levels of several proteins of the mitochondrial complex III and its activity in the mouse liver

Ascorbate is a crucial antioxidant and essential cofactor of biosynthetic and regulatory enzymes. Unlike humans, mice can synthesize ascorbate thanks to the key enzyme gulonolactone oxidase (Gulo). In the present study, we used the Gulo−/− mouse model, which cannot synthesize their own ascorbate to determine the impact of this vitamin on the liver proteome of specific subcellular organelles. We performed label-free Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) global quantitative proteomic profiling to identify and quantify proteins in microsomal enriched liver extracts (MEE) from Gulo−/− mice treated with 0–0.4% (w/v) ascorbate in drinking water until the age of four months. Using a principal component analysis on normalized and imputed data of the label-free protein quantifications, a sex-based difference in MEE proteome profiles was observed for all the different ascorbate treated mice. Suboptimal hepatic ascorbate concentrations affected the levels of more proteins and hence biochemical processes in females than in males. Nevertheless, Pearson correlation analyses revealed that the MS intensities of various proteins involved in complement activation inversely correlated with liver ascorbate concentrations in both Gulo−/− males and females. Moreover, the correlation analyses also indicated that several proteins in the mitochondrial complex III of the electron transport chain positively correlated with liver ascorbate concentrations in both Gulo−/− females and males. Consequently, the mitochondrial complex III activity in Gulo−/− female and male mice treated with suboptimal hepatic concentrations of ascorbate was significantly lower than Gulo−/− mice treated with optimal ascorbate concentration. Finally, the whole liver of ascorbate-deficient Gulo−/− mice exhibited lower ATP levels and increased reactive oxygen species. These findings provide new information on how ascorbate deficiency potentially induces mitochondrial dysfunction in the liver of mice.

Screening membraneless organelle participants with machine-learning models that integrate multimodal features

Protein self-assembly is one of the formation mechanisms of biomolecular condensates. However, most phase-separating systems (PS) demand multiple partners in biological conditions. In this study, we divided PS proteins into two groups according to the mechanism by which they undergo PS: PS-Self proteins can self-assemble spontaneously to form droplets, while PS-Part proteins interact with partners to undergo PS. Analysis of the amino acid composition revealed differences in the sequence pattern between the two protein groups. Existing PS predictors, when evaluated on two test protein sets, preferentially predicted self-assembling proteins. Thus, a comprehensive predictor is required. Herein, we propose that properties other than sequence composition can provide crucial information in screening PS proteins. By incorporating phosphorylation frequencies and immunofluorescence image-based droplet-forming propensity with other PS-related features, we built two independent machine-learning models to separately predict the two protein categories. Results of independent testing suggested the superiority of integrating multimodal features. We performed experimental verification on the top-scored proteins DHX9, Ki-67, and NIFK. Their PS behavior invitro revealed the effectiveness of our models in PS prediction. Further validation on the proteome of membraneless organelles confirmed the ability of our models to identify PS-Part proteins. We implemented a web server named PhaSePred (http://predict.phasep.pro/) that incorporates our two models together with representative PS predictors. PhaSePred displays proteome-level quantiles of different features, thus profiling PS propensity and providing crucial information for identification of candidate proteins.

Mitochondrial сomplexome of etiolated pea shoots

Studies into mitochondrial сomplexomes in various organisms provide an insight into the native organization of proteins and metabolic pathways in the organelles of the subject under study. “Complexome” is a relatively recent concept describing the proteome of protein complexes, supercomplexes, and oligomeric proteins. Complexome analysis is performed using current electrophoretic and mass spectrometric techniques, in particular, by two-dimensional electrophoresis (2D BN/SDS-PAGE) in combination with mass spectrometry (MS). Unlike 2D IEF/SDS-PAGE, this method enables analysis of not only hydrophilic proteins of the mitochondrial matrix, but also membrane proteins and their associations, thus expanding the possibilities of studying the organelle proteome. In the present work, the complexome of etiolated pea shoots was studied for the first time using 2D BN/SDS-PAGE followed by MALDI-TOF MS. To this end, 145 protein spots excised from the gel were analyzed; 110 polypeptides were identified and assigned to different functional groups. A densitometric analysis revealed that the major protein group comprised the enzymes of the mitochondrial energy system (1), accounting for an average of 43% of the total polypeptide content. The remaining 57% was primarily distributed among the following functional categories: pyruvate dehydrogenase complex and citric acid cycle (2); amino acid metabolism (3); nucleic acid processing (4); protein folding (5); antioxidant protection (6); carrier proteins (7); other proteins (8); proteins having unknown functions (9). The obtained data indicate the complex organization of the pea proteome. In addition to the enzymes of the OXPHOS system, the proteins of other functional categories are found to form supramolecular structures. It is suggested that the presence of proteins from other cellular compartments may indicate the interaction of mitochondria with the enzymes or structures of corresponding organelles. In general, the obtained data on the pea complexome represent a kind of a mitochondrial “passport” that reflects the native state of the proteome of organelles corresponding to their physiological status.

Phosphoproteomics and Organelle Proteomics in Pancreatic Islets.

Over the recent years, mass spectrometry (MS)-based proteomics has undergone dramatic advances in sample preparation, instrumentation, and computational methods. Here, we describe in detail, how a workflow quantifies global protein phosphorylation in pancreatic islets and characterizes intracellular organelle composition on protein level by MS-based proteomics.

Biotinylation-based proximity labelling proteomics: basics, applications and technical considerations.

Recent advances in biotinylation-based proximity labelling (PL) have opened up new avenues for mapping the protein composition of cellular compartments and protein complexes in living cells at high spatiotemporal resolution. In particular, PL combined with mass spectrometry-based proteomics has been successfully applied to defining protein-protein interactions, protein-nucleic acid interactions, (membraneless) organelle proteomes and secretomes in various systems ranging from cultured cells to whole animals. In this review, we first summarize the basics and recent biological applications of PL proteomics and then highlight recent developments in enrichment techniques for biotinylated proteins and peptides, focusing on the advantages of PL and technical considerations.

Human genetic analyses of organelles highlight the nucleus in age-related trait heritability.

Most age-related human diseases are accompanied by a decline in cellular organelle integrity, including impaired lysosomal proteostasis and defective mitochondrial oxidative phosphorylation. An open question, however, is the degree to which inherited variation in or near genes encoding each organelle contributes to age-related disease pathogenesis. Here, we evaluate if genetic loci encoding organelle proteomes confer greater-than-expected age-related disease risk. As mitochondrial dysfunction is a 'hallmark' of aging, we begin by assessing nuclear and mitochondrial DNA loci near genes encoding the mitochondrial proteome and surprisingly observe a lack of enrichment across 24 age-related traits. Within nine other organelles, we find no enrichment with one exception: the nucleus, where enrichment emanates from nuclear transcription factors. In agreement, we find that genes encoding several organelles tend to be 'haplosufficient,' while we observe strong purifying selection against heterozygous protein-truncating variants impacting the nucleus. Our work identifies common variation near transcription factors as having outsize influence on age-related trait risk, motivating future efforts to determine if and how this inherited variation then contributes to observed age-related organelle deterioration.

A modified density gradient proteomic-based method to analyze endolysosomal proteins in cardiac tissue

SummaryThe importance of lysosomes in cardiac physiology and pathology is well established, and evidence for roles in calcium signaling is emerging. We describe a label-free proteomics method suitable for small cardiac tissue biopsies based on density-separated fractionation, which allows study of endolysosomal (EL) proteins. Density gradient fractions corresponding to tissue lysate; sarcoplasmic reticulum (SR), mitochondria (Mito) (1.3 g/mL); and EL with negligible contamination from SR or Mito (1.04 g/mL) were analyzed using Western blot, enzyme activity assay, and liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis (adapted discontinuous Percoll and sucrose differential density gradient). Kyoto Encyclopedia of Genes and Genomes, Reactome, Panther, and Gene Ontology pathway analysis showed good coverage of RAB proteins and lysosomal cathepsins (including cardiac-specific cathepsin D) in the purified EL fraction. Significant EL proteins recovered included catalytic activity proteins. We thus present a comprehensive protocol and data set of guinea pig atrial EL organelle proteomics using techniques also applicable for non-cardiac tissue.

Proteomic Identification of Bombyx mori Organelles Using the Engineered Ascorbate Peroxidase APEX and Development of Silkworm Organelle Proteome Database (SilkOrganPDB).

Silkworm Bombyx mori is an economically important insect and a lepidopteran model. Organelle proteome is vital to understanding gene functions; however, it remains to be identified in silkworm. Here, using the engineered ascorbate peroxidase APEX, we constructed transgenic B. mori embryo cells (BmE) expressing APEX-NLS, COX4-APEX, APEX-Rev, and APEX-KDEL in nucleus, mitochondrial matrix (MM), cytosol, and endoplasmic reticulum (ER), and isolated the biotin-labeled proteins using streptavidin-affinity purification, respectively. The isolated proteins were determined using LC-MS/MS and annotated by searching B. mori genomes downloaded from GenBank, SilkBase, SilkDB 2.0, and SilkDB 3.0, resulting in 842, 495, 311, and 445 organelle proteins identified, respectively. We mapped the 296 MM proteins annotated in the GenBank data to mitochondrial protein databases of the fly, human, and mouse, and found that 140 (47%) proteins are homologous to 80 fly proteins, and 65 (22%) proteins match to 31 and 29 human and mouse proteins, respectively. Protein orthology was predicted in multiple insects using OrthoMCL, producing 460 families containing 839 proteins we identified. Out of 460 families, 363 were highly conserved and found in all insects, leaving only three proteins without orthology in other insects, indicating that the identified proteins are highly conserved and probably play important roles in insects. A gene ontology enrichment analysis by clusterProfiler revealed that the nucleus proteins significantly enriched in cellular component terms of nucleus and nucleolus, the MM proteins markedly enriched in molecular function terms of nucleotide binding, and the cytosol proteins mainly enriched in biological process terms of small molecule metabolism. To facilitate the usage and analysis of our data, we developed an open-access database, Silkworm Organelle Proteome Database (SilkOrganPDB), which provides multiple modules for searching, browsing, downloading, and analyzing these proteins, including BLAST, HMMER, Organelle Proteins, Protein Locations, Sequences, Gene Ontology, Homologs, and Phylogeny. In summary, our work revealed the protein composition of silkworm BmE organelles and provided a database resource helpful for understanding the functions and evolution of these proteins.

Proximity‐Dependent Sensors Reveal New Mechanisms of mTORC1 Activation by Amino Acids

The mechanistic Target of Rapamycin Complex 1 (mTORC1) is potently activated by amino acids (AAs), which may be acquired exogenously through cell surface transporters, or derived through lysosomal degradation of exogenous protein. In highly lethal Ras-driven cancers, macropinocytosis and lysosomal degradation of exogenous protein has been shown to fuel cancer growth through unclear mechanisms. Both exogenous and lysosome-derived AAs activate mTORC1 on the lysosome surface. We hypothesized that detailed characterization of late endosome and lysosome organelle proteomes would reveal the functional organization of mTORC1 regulatory machinery, offering potential insight into mTORC1 activation by distinct sources of AAs. Using proximity-dependent biotinylation and mass spectrometry (BioID), we designed organelle ‘sensors’ with which we revealed the surface proteomes of late endosomes and lysosomes. By combining BioID ‘sensors’ with systematic gene ablation using CRISPR-Cas9 (a technique we call KO-BioID) we were able to further define the functional organization of key proteins and complexes within the late endocytic system. Our results demonstrated that mTORC1 regulatory machinery is targeted to a specific subdomain of the late endocytic system that is defined by the HOPS complex. Functionally, we demonstrated that mTORC1 activation by lysosome-derived AAs operates through a Rag GTPase-independent pathway, which is inhibited by activation of the GATOR-Rag GTPase AA-sensing pathway. In summary, our work shows that distinct but functionally opposed mechanisms exist to activate mTORC1 in response to different AA sources. These results may reveal mechanistic insight into how lysosome-derived nutrients fuel growth of Ras-driven cancers.