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The SLC35 (Solute Carrier 35) family members acting as nucleotide sugar transporters are typically localized in the endoplasmic reticulum or Golgi apparatus. It is, therefore, intriguing that some reports document the presence of orphan transporters SLC35F1 and SLC35F6 within the endosomal and lysosomal system. Here, we compared the subcellular distribution of these proteins and found that they are concentrated in separate compartments; i.e., recycling endosomes for SLC35F1 and lysosomes for SLC35F6. Swapping the C-terminal tail of these proteins resulted in a switch of localization, with SLC35F1 being trafficked to lysosomes while SLC35F6 remained in endosomes. This suggested the presence of specific sorting signals in these C-terminal regions. Using site-directed mutagenesis, fluorescence microscopy, and cell surface biotinylation assays, we found that the EQERLL 360 signal located in the cytoplasmic tail of human SLC35F6 is involved in its lysosomal sorting (as previously shown for this conserved sequence in mouse SLC35F6), and that SLC35F1 localization in the recycling pathway depends on two YXXΦ-type signals: a Y 367 KQF sequence facilitates its internalization from the plasma membrane, while a Y 392 TSL motif prevents its transport to lysosomes, likely by promoting SLC35F1 recycling to the cell surface. Taken together, these results support that some SLC35 members may function at different levels of the endosomal and lysosomal system.

The facultative intracellular pathogen Brucella abortus interacts with several organelles of the host cell to reach its replicative niche inside the endoplasmic reticulum. However, little is known about the interplay between the intracellular bacteria and the host cell mitochondria. Here, we showed that B. abortus triggers substantive mitochondrial network fragmentation, accompanied by mitophagy and the formation of mitochondrial Brucella-containing vacuoles during the late steps of cellular infection. Brucella-induced expression of the mitophagy receptor BNIP3L is essential for these events and relies on the iron-dependent stabilisation of the hypoxia-inducible factor 1α. Functionally, BNIP3L-mediated mitophagy appears to be advantageous for bacterial exit from the host cell as BNIP3L depletion drastically reduces the number of reinfection events. Altogether, these findings highlight the intricate link between Brucella trafficking and the mitochondria during host cell infection., (© 2023 The Authors.)

Mucopolysaccharidosis IX is a lysosomal storage disorder caused by a deficiency in HYAL1, an enzyme that degrades hyaluronic acid at acidic pH. This disease causes juvenile arthritis in humans and osteoarthritis in the Hyal1 knockout mouse model. Our past research revealed that HYAL1 is strikingly upregulated (~ 25x) upon differentiation of bone marrow monocytes into osteoclasts. To investigate whether HYAL1 is involved in the differentiation and/or resorption activity of osteoclasts, and in bone remodeling in general, we analyzed several bone parameters in Hyal1 -/- mice and studied the differentiation and activity of their osteoclasts and osteoblasts when differentiated in vitro. These experiments revealed that, upon aging, HYAL1 deficient mice exhibit reduced femur length and a ~ 15% decrease in bone mineral density compared to wild-type mice. We found elevated osteoclast numbers in the femurs of these mice as well as an increase of the bone resorbing activity of Hyal1 -/- osteoclasts. Moreover, we detected decreased mineralization by Hyal1 -/- osteoblasts. Taken together with the observed accumulation of hyaluronic acid in Hyal1 -/- bones, these results support the premise that the catabolism of hyaluronic acid by osteoclasts and osteoblasts is an intrinsic part of bone remodeling., (© 2022. The Author(s).)

Knowledge of intracellular location can provide important insights into the function of proteins and their respective organelles, and there is interest in combining classical subcellular fractionation with quantitative mass spectrometry to create global cellular maps. To evaluate mass spectrometric approaches specifically for this application, we analyzed rat liver differential centrifugation and Nycodenz density gradient subcellular fractions by tandem mass tag (TMT) isobaric labeling with reporter ion measurement at the MS2 and MS3 level and with two different label-free peak integration approaches, MS1 and data independent acquisition (DIA). TMT-MS2 provided the greatest proteome coverage, but ratio compression from contaminating background ions resulted in a narrower accurate dynamic range compared to TMT-MS3, MS1, and DIA, which were similar. Using a protein clustering approach to evaluate data quality by assignment of reference proteins to their correct compartments, all methods performed well, with isobaric labeling approaches providing the highest quality localization. Finally, TMT-MS2 gave the lowest percentage of missing quantifiable data when analyzing orthogonal fractionation methods containing overlapping proteomes. In summary, despite inaccuracies resulting from ratio compression, data obtained by TMT-MS2 assigned protein localization as well as other methods but achieved the highest proteome coverage with the lowest proportion of missing values.

Obesity is characterized by an excessive triacylglycerol accumulation in white adipocytes. Various mechanisms allowing the tight regulation of triacylglycerol storage and mobilization by lipid droplet-associated proteins as well as lipolytic enzymes have been identified. Increasing energy expenditure by inducing a mild uncoupling of mitochondria in adipocytes might represent a putative interesting anti-obesity strategy as it reduces the adipose tissue triacylglycerol content (limiting alterations caused by cell hypertrophy) by stimulating lipolysis through yet unknown mechanisms, limiting the adverse effects of adipocyte hypertrophy. Herein, the molecular mechanisms involved in lipolysis induced by a mild uncoupling of mitochondria in white 3T3-L1 adipocytes were characterized. Mitochondrial uncoupling-induced lipolysis was found to be independent from canonical pathways that involve lipolytic enzymes such as HSL and ATGL. Finally, enhanced lipolysis in response to mitochondrial uncoupling relies on a form of autophagy as lipid droplets are captured by endolysosomal vesicles. This new mechanism of triacylglycerol breakdown in adipocytes exposed to mild uncoupling provides new insights on the biology of adipocytes dealing with mitochondria forced to dissipate energy., (© 2017 Wiley Periodicals, Inc.)

Lysosomes degrade cellular components sequestered by autophagy or extracellular material internalized by endocytosis and phagocytosis. The macromolecule building blocks released by lysosomal hydrolysis are then exported to the cytosol by lysosomal transporters, which remain undercharacterized. In this study, we designed an in situ assay of lysosomal amino acid export based on the transcription factor EB (TFEB), a master regulator of lysosomal biogenesis that detects lysosomal storage. This assay was used to screen candidate lysosomal transporters, leading to the identification of sodium-coupled neutral amino acid transporter 7 (SNAT7), encoded by the SLC38A7 gene, as a lysosomal transporter highly selective for glutamine and asparagine. Cell fractionation confirmed the lysosomal localization of SNAT7, and flux measurements confirmed its substrate selectivity and showed a strong activation by the lysosomal pH gradient. Interestingly, gene silencing or editing experiments revealed that SNAT7 is the primary permeation pathway for glutamine across the lysosomal membrane and it is required for growth of cancer cells in a low free-glutamine environment, when macropinocytosis and lysosomal degradation of extracellular proteins are used as an alternative source of amino acids. SNAT7 may, thus, represent a novel target for glutamine-related anticancer therapies., Competing Interests: Conflict of interest statement: The authors have filed a European patent application on potential therapeutic uses of SNAT7-directed tools.

Accurate knowledge of the intracellular location of proteins is important for numerous areas of biomedical research including assessing fidelity of putative protein-protein interactions, modeling cellular processes at a system-wide level and investigating metabolic and disease pathways. Many proteins have not been localized, or have been incompletely localized, partly because most studies do not account for entire subcellular distribution. Thus, proteins are frequently assigned to one organelle whereas a significant fraction may reside elsewhere. As a step toward a comprehensive cellular map, we used subcellular fractionation with classic balance sheet analysis and isobaric labeling/quantitative mass spectrometry to assign locations to >6000 rat liver proteins. We provide quantitative data and error estimates describing the distribution of each protein among the eight major cellular compartments: nucleus, mitochondria, lysosomes, peroxisomes, endoplasmic reticulum, Golgi, plasma membrane and cytosol. Accounting for total intracellular distribution improves quality of organelle assignments and assigns proteins with multiple locations. Protein assignments and supporting data are available online through the Prolocate website (http://prolocate.cabm.rutgers.edu). As an example of the utility of this data set, we have used organelle assignments to help analyze whole exome sequencing data from an infant dying at 6 months of age from a suspected neurodegenerative lysosomal storage disorder of unknown etiology. Sequencing data was prioritized using lists of lysosomal proteins comprising well-established residents of this organelle as well as novel candidates identified in this study. The latter included copper transporter 1, encoded by SLC31A1, which we localized to both the plasma membrane and lysosome. The patient harbors two predicted loss of function mutations in SLC31A1, suggesting that this may represent a heretofore undescribed recessive lysosomal storage disease gene., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

ATG9A is the only polytopic protein of the mammalian autophagy-related protein family whose members regulate autophagosome formation during macroautophagy. At steady state, ATG9A localizes to several intracellular sites, including the Golgi apparatus, endosomes and the plasma membrane, and it redistributes towards autophagosomes upon autophagy induction. Interestingly, the transport of yeast Atg9 to the pre-autophagosomal structure depends on its self-association, which is mediated by a short amino acid motif located in the C-terminal region of the protein. Here, we investigated whether the residues that align with this motif in human ATG9A (V 515 -C 519 ) are also required for its trafficking in mammalian cells. Interestingly, our findings support that human ATG9A self-interacts as well, and that this process promotes transport of ATG9A molecules through the Golgi apparatus. Furthermore, our data reveal that the transport of ATG9A out of the ER is severely impacted after mutation of the conserved V 515 -C 519 motif. Nevertheless, the mutated ATG9A molecules could still interact with each other, indicating that the molecular mechanism of self-interaction differs in mammalian cells compared to yeast. Using sequential amino acid substitutions of glycine 516 and cysteine 519, we found that the stability of ATG9A relies on both of these residues, but that only the former is required for efficient transport of human ATG9A from the endoplasmic reticulum to the Golgi apparatus., (Copyright © 2016 Elsevier Inc. All rights reserved.)

ATG9A is a multispanning membrane protein required for autophagosome formation. Under basal conditions, neosynthesized ATG9A proteins travel to the Golgi apparatus and cycle between the trans-Golgi network and endosomes. In the present work, we searched for molecular determinants involved in the subcellular trafficking of human ATG9A in HeLa cells using sequential deletions and point mutations. Deletion of amino acids L(340) to L(354) resulted in the retention of ATG9A in the endoplasmic reticulum. In addition, we found that substitution of the L(711)YM(713) sequence (located in the C-terminal region of ATG9A) by alanine residues severely impaired its transport through the Golgi apparatus. This defect could be corrected by oligomerization of the mutant protein with co-transfected wild-type ATG9A, suggesting that ATG9A oligomerization may help its sorting through biosynthetic compartments. Lastly, the study of the consequences of the LYM/AAA mutation on the intracellular trafficking of ATG9A highlighted that some newly synthesized ATG9A can bypass the Golgi apparatus to reach the plasma membrane. Taken together, these findings provide new insights into the intracellular pathways followed by ATG9A to reach different subcellular compartments, and into the intramolecular determinants that drive the sorting of this protein., (Copyright © 2016 Elsevier B.V. All rights reserved.)

How, in the absence of a functional mannose 6-phosphate (Man-6-P)-signal-dependent transport pathway, some acid hydrolases remain sorted to endolysosomes in the brain is poorly understood. We demonstrate that cathepsin D binds to mouse SEZ6L2, a type 1 transmembrane protein predominantly expressed in the brain. Studies of the subcellular trafficking of SEZ6L2, and its silencing in a mouse neuroblastoma cell line reveal that SEZ6L2 is involved in the trafficking of cathepsin D to endosomes. Moreover, SEZ6L2 can partially correct the cathepsin D hypersecretion resulting from the knockdown of UDP-GlcNAc:lysosomal enzyme GlcNAc-1-phosphotransferase in HeLa cells (i.e. in cells that are unable to synthesize Man-6-P signals). Interestingly, cleavage of SEZ6L2 by cathepsin D generates an N-terminal soluble fragment that induces neurite outgrowth, whereas its membrane counterpart prevents this. Taken together, our findings highlight that SEZ6L2 can serve as receptor to mediate the sorting of cathepsin D to endosomes, and suggest that proteolytic cleavage of SEZ6L2 by cathepsin D modulates neuronal differentiation., (© 2016. Published by The Company of Biologists Ltd.)

The Na(+)/K(+)-ATPase interacts with the non-selective cation channels TRPC6 but the functional consequences of this association are unknown. Experiments performed with HEK cells over-expressing TRPC6 channels showed that inhibiting the activity of the Na(+)/K(+)-ATPase with ouabain reduced the amount of TRPC6 proteins and depressed Ca(2+) entry through TRPC6. This effect, not mimicked by membrane depolarization with KCl, was abolished by sucrose and bafilomycin-A, and was partially sensitive to the intracellular Ca(2+) chelator BAPTA/AM. Biotinylation and subcellular fractionation experiments showed that ouabain caused a multifaceted redistribution of TRPC6 to the plasma membrane and to an endo/lysosomal compartment where they were degraded. The amyloid beta peptide Aβ(1-40), another inhibitor of the Na(+)/K(+)-ATPase, but not the shorter peptide Aβ1-16, reduced TRPC6 protein levels and depressed TRPC6-mediated responses. In cortical neurons from embryonic mice, ouabain, veratridine (an opener of voltage-gated Na(+) channel), and Aβ(1-40) reduced TRPC6-mediated Ca(2+) responses whereas Aβ(1-16) was ineffective. Furthermore, when Aβ(1-40) was co-added together with zinc acetate it could no longer control TRPC6 activity. Altogether, this work shows the existence of a functional coupling between the Na(+)/K(+)-ATPase and TRPC6. It also suggests that the abundance, distribution and activity of TRPC6 can be regulated by cardiotonic steroids like ouabain and the naturally occurring peptide Aβ(1-40) which underlines the pathophysiological significance of these processes., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: Several intracellular bacterial pathogens have evolved subtle strategies to subvert vesicular trafficking pathways of their host cells to avoid killing and to replicate inside the cells. Brucellae are Gram-negative facultative intracellular bacteria that are responsible for brucellosis, a worldwide extended chronic zoonosis. Following invasion, Brucella abortus is found in a vacuole that interacts first with various endosomal compartments and then with endoplasmic reticulum sub-compartments. Brucella establishes its replication niche in ER-derived vesicles. In the past, it has been proposed that B. abortus passed through the macroautophagy pathway before reaching its niche of replication. However, recent experiments provided evidence that the classical macroautophagy pathway was not involved in the intracellular trafficking and the replication of B. abortus in bone marrow-derived macrophages and in HeLa cells. In contrast, another study showed that macroautophagy favoured the survival and the replication of Brucella melitensis in infected RAW264.7 macrophages. This raises the possibility that B. abortus and B. melitensis followed different intracellular pathways before replicating. In the present work, we have addressed this issue by comparing the replication rate of B. abortus and B. melitensis in embryonic fibroblasts derived from wild-type and Atg5-/- mice, Atg5 being a core component of the canonical macroautophagic pathway., Results: Our results indicate that both B. abortus S2308 and B. melitensis 16M strains are able to invade and replicate in Atg5-deficient fibroblasts, suggesting that the canonical Atg5-dependent macroautophagic pathway is dispensable for Brucella replication. The number of viable bacteria was even slightly higher in Atg5-/- fibroblasts than in wild-type fibroblasts. This increase could be due to a more efficient uptake or to a better survival rate of bacteria before the beginning of the replication in Atg5-deficient cells as compared to wild-type cells. Moreover, our data show that the infection with B. abortus or with B. melitensis does not stimulate neither the conversion of LC3-I to LC3-II nor the membrane recruitment of LC3 onto the BCV., Conclusion: Our study suggests that like Brucella abortus, Brucella melitensis does not subvert the canonical macroautophagy to reach its replicative niche or to stimulate its replication.

The hyaluronidase Hyal-1 is an acid hydrolase that degrades hyaluronic acid (HA), a component of the extracellular matrix. It is often designated as a lysosomal protein. Yet few data are available on its intracellular localization and trafficking. We demonstrate here that in RAW264.7 murine macrophages, Hyal-1 is synthesized as a glycosylated precursor that is only weakly mannose 6-phosphorylated. Nevertheless, this precursor traffics to endosomes, via a mannose 6-phosphate-independent secretion/recapture mechanism that involves the mannose receptor. Once in endosomes, it is processed into a lower molecular mass form that is transported to lysosomes, where its activity could be detected using native gel zymography. Indeed, this activity co-distributed with lysosomal hydrolases in the densest fraction of a self-forming Percoll(TM) density gradient. Moreover, it shifted toward the lower density region, in parallel with those hydrolases, when a decrease of lysosomal density was induced by the endocytosis of sucrose. Interestingly, the activity of the processed form of Hyal-1 was largely underestimated when assayed by zymography after SDS-PAGE and subsequent renaturation of the proteins, by contrast to the full-length protein that could efficiently degrade HA in those conditions. These results suggest that noncovalent associations support the lysosomal activity of Hyal-1., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

It has long been known that liver lysosomes contain an endoglycosidase activity able to degrade the high molecular mass glycosaminoglycan hyaluronic acid (HA). The identification and cloning of a hyaluronidase with an acidic pH optimum, Hyal-1, suggested it might be responsible for this activity. However, we previously reported that this hydrolase could only be detected in pre-lysosomal compartments of the mouse liver using a zymography technique that allows the detection of Hyal-1 activity after SDS-PAGE ("renatured protein zymography"). Present work reveals that the activity highlighted by this technique belongs to a precursor form of Hyal-1 and that the lysosomal HA endoglycosidase activity of the mouse liver is accounted for by a proteolytically processed form of Hyal-1 that can only be detected using "native protein zymography". Indeed, the distribution of this form follows the distribution of β-galactosidase, a well-established lysosomal marker, after fractionation of the mouse liver in a linear sucrose density gradient. In addition, both activities shift toward the lower density region of the gradient when a specific decrease of the lysosomal density is induced by Triton WR-1339 injection. The fact that only native protein zymography but not renatured protein zymography is able to detect Hyal-1 activity in lysosomes points to a non-covalent association of Hyal-1 proteolytic fragments or the existence of closely linked partners supporting Hyal-1 enzymatic activity. The knockdown of Hyal-1 results in an 80% decrease of total acid hyaluronidase activity in the mouse liver, confirming that Hyal-1 is a key actor of HA catabolism in this organ., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Lysosomes are membrane-bound endocytic organelles that play a major role in degrading cell macromolecules and recycling their building blocks. A comprehensive knowledge of the lysosome function requires an extensive description of its content, an issue partially addressed by previous proteomic analyses. However, the proteins underlying many lysosomal membrane functions, including numerous membrane transporters, remain unidentified. We performed a comparative, semi-quantitative proteomic analysis of rat liver lysosome-enriched and lysosome-nonenriched membranes and used spectral counts to evaluate the relative abundance of proteins. Among a total of 2,385 identified proteins, 734 proteins were significantly enriched in the lysosomal fraction, including 207 proteins already known or predicted as endo-lysosomal and 94 proteins without any known or predicted subcellular localization. The remaining 433 proteins had been previously assigned to other subcellular compartments but may in fact reside on lysosomes either predominantly or as a secondary location. Many membrane-associated complexes implicated in diverse processes such as degradation, membrane trafficking, lysosome biogenesis, lysosome acidification, signaling, and nutrient sensing were enriched in the lysosomal fraction. They were identified to an unprecedented extent as most, if not all, of their subunits were found and retained by our screen. Numerous transporters were also identified, including 46 novel potentially lysosomal proteins. We expressed 12 candidates in HeLa cells and observed that most of them colocalized with the lysosomal marker LAMP1, thus confirming their lysosomal residency. This list of candidate lysosomal proteins substantially increases our knowledge of the lysosomal membrane and provides a basis for further characterization of lysosomal functions.

NCLs (neuronal ceroid lipofuscinoses) form a group of eight inherited autosomal recessive diseases characterized by the intralysosomal accumulation of autofluorescent pigments, called ceroids. Recent data suggest that the pathogenesis of NCL is associated with the appearance of fragmented mitochondria with altered functions. However, even if an impairement in the autophagic pathway has often been evoked, the molecular mechanisms leading to mitochondrial fragmentation in response to a lysosomal dysfunction are still poorly understood. In this study, we show that fibroblasts that are deficient for the TPP-1 (tripeptidyl peptidase-1), a lysosomal hydrolase encoded by the gene mutated in the LINCL (late infantile NCL, CLN2 form) also exhibit a fragmented mitochondrial network. This morphological alteration is accompanied by an increase in the expression of the protein BNIP3 (Bcl2/adenovirus E1B 19 kDa interacting protein 3) as well as a decrease in the abundance of mitofusins 1 and 2, two proteins involved in mitochondrial fusion. Using RNAi (RNA interference) and quantitative analysis of the mitochondrial morphology, we show that the inhibition of BNIP3 expression does not result in an increase in the reticulation of the mitochondrial population in LINCL cells. However, this protein seems to play a key role in cell response to mitochondrial oxidative stress as it sensitizes mitochondria to antimycin A-induced fragmentation. To our knowledge, our results bring the first evidence of a mechanism that links TPP-1 deficiency and oxidative stress-induced changes in mitochondrial morphology.

Lysosomes are cytoplasmic organelles delimited by a single membrane and filled with a variety of hydrolytic enzymes active at acidic pH and collectively capable to degrade the vast majority of macromolecules entering lysosomes via endocytosis, phagocytosis or autophagy. In this review, we describe the lipid composition and the dynamic properties of lysosomal membrane, the main delivery pathways of lipids to lysosomes and their catabolism inside lysosomes. Then, we present the consequences of a lipid accumulation as seen in various lysosomal storage diseases on lysosomal functions. Finally, we discuss about the possible involvement of lysosomes in lipotoxicity.

The rapid turnover rate of hyaluronan (HA), the major unbranched glycosaminoglycan of the extracellular matrix, is dependent on hyaluronidases. One of them, hyaluronidase-2 (Hyal2), degrades HA into smaller fragments endowed with specific biological activities such as inflammation and angiogenesis. Yet the cellular environment of Hyal2, a purported glycosylphosphatidylinositol (GPI)-anchored protein, remains uncertain. We have examined the membrane association of Hyal2 in MDA-MB231 cancer cells where it is highly expressed and in COS-7 cells transfected with native or fluorescent Hyal2 constructs. In both cell types, Hyal2 was strongly associated with cell membrane fractions from which it could be extracted using a Triton X-114 treatment (hydrophobic phase) but not an osmotic shock or an alkaline carbonate solution. Treatment of membrane preparations with phosphatidylinositol-specific phospholipase C released immunoreactive Hyal2 into the aqueous phase, confirming the protein is attached to the membrane through a functional GPI anchor. Hyal2 transfected in COS-7 cells was associated with detergent-resistant, cholesterol-rich membranes known as lipid rafts. The cellular immunofluorescent pattern of Hyal2 was conditioned by the presence of a GPI anchor. In summary, the strong membrane association of Hyal2 through its GPI anchor demonstrated in this study using biochemical methods suggests that the main activity of this enzyme is located at the level of the plasma membrane in close contact with the pericellular HA-rich glycocalyx, the extracellular matrix, or possibly endocytic vesicles., (Copyright © 2011 Elsevier Inc. All rights reserved.)

One approach to the functional characterization of the lysosome lies in the use of proteomic methods to identify proteins in subcellular fractions enriched for this organelle. However, distinguishing between true lysosomal residents and proteins from other cofractionating organelles is challenging. To this end, we implemented a quantitative mass spectrometry approach based on the selective decrease in the buoyant density of liver lysosomes that occurs when animals are treated with Triton-WR1339. Liver lysosome-enriched preparations from control and treated rats were fractionated by isopycnic sucrose density gradient centrifugation. Tryptic peptides derived from gradient fractions were reacted with isobaric tag for relative and absolute quantitation eight-plex labeling reagents and analyzed by two-dimensional liquid chromatography matrix-assisted laser desorption ionization time-of-flight MS. Reporter ion intensities were used to generate relative protein distribution profiles across both types of gradients. A distribution index was calculated for each identified protein and used to determine a probability of lysosomal residence by quadratic discriminant analysis. This analysis suggests that several proteins assigned to the lysosome in other proteomics studies are not true lysosomal residents. Conversely, results support lysosomal residency for other proteins that are either not or only tentatively assigned to this location. The density shift for two proteins, Cu/Zn superoxide dismutase and ATP-binding cassette subfamily B (MDR/TAP) member 6, was corroborated by quantitative Western blotting. Additional balance sheet analyses on differential centrifugation fractions revealed that Cu/Zn superoxide dismutase is predominantly cytosolic with a secondary lysosomal localization whereas ATP-binding cassette subfamily B (MDR/TAP) member 6 is predominantly lysosomal. These results establish a quantitative mass spectrometric/subcellular fractionation approach for identification of lysosomal proteins and underscore the necessity of balance sheet analysis for localization studies.

Niemann-Pick Type C (NPC) disease is a lysosomal storage disorder characterized by accumulation of unesterified cholesterol and other lipids in the endolysosomal system. NPC disease results from a defect in either of two distinct cholesterol-binding proteins: a transmembrane protein, NPC1, and a small soluble protein, NPC2. NPC1 and NPC2 are thought to function closely in the export of lysosomal cholesterol with both proteins binding cholesterol in vitro but they may have unrelated lysosomal roles. To investigate this possibility, we compared biochemical consequences of the loss of either protein. Analyses of lysosome-enriched subcellular fractions from brain and liver revealed similar decreases in buoyant densities of lysosomes from NPC1 or NPC2 deficient mice compared to controls. The subcellular distribution of both proteins was similar and paralleled a lysosomal marker. In liver, absence of either NPC1 or NPC2 resulted in similar alterations in the carbohydrate processing of the lysosomal protease, tripeptidyl peptidase I. These results highlight biochemical alterations in the lysosomal system of the NPC-mutant mice that appear secondary to lipid storage. In addition, the similarity in biochemical phenotypes resulting from either NPC1 or NPC2 deficiency supports models in which the function of these two proteins within lysosomes are linked closely.

It has been suggested that intracellular Hyal-1 (hyaluronidase-1), which is considered a lysosomal enzyme, originates via endocytosis of the serum enzyme. To test this proposal we have investigated the uptake and intracellular distribution of rhHyal-1 (recombinant human Hyal-1) by mouse liver, making use of centrifugation methods. Experiments were performed on wild-type mice injected with 125I-labelled rhHyal-1 and on Hyal-1-/- mice injected with the unlabelled enzyme, which were killed at various times after injection. Activity of the unlabelled enzyme was determined by zymography. Intracellular distribution of Hyal-1 was investigated by differential and isopycnic centrifugation. The results of the study indicated that rhHyal-1 is endocytosed by the liver, mainly by sinusoidal cells, and follows the intracellular pathway described for many endocytosed proteins that are eventually located in lysosomes. However, Hyal-1 endocytosis has some particular features. First, endocytosed rhHyal-1 is quickly degraded. Secondly, its distribution, as analysed by differential centrifugation, differs from the distribution of beta-galactosidase, taken as the reference lysosomal enzyme. Further analysis by isopycnic centrifugation in a sucrose gradient shows endocytosed rhHyal-1 behaves like beta-galactosidase shortly after injection. However the Hyal-1 distribution is markedly less affected than beta-galactosidase, following a prior injection of Triton WR-1339, which is a specific density perturbant of lysosomes. The behaviour in centrifugation of endogenous liver Hyal-1, identified by hyaluronan zymography, exhibits some similarity with the behaviour of the endocytosed enzyme, suggesting that it could originate from endocytosis of the serum enzyme. Overall, these results can be explained by supposing that active endocytosed Hyal-1 is mainly present in early lysosomes. Although its degradation half-time is short, Hyal-1 could exert its activity due to a constant supply of active molecules from the blood.

Classical late-infantile neuronal ceroid lipofuscinosis (LINCL) is a fatal neurodegenerative disease of children caused by mutations in TPP1, the gene encoding the lysosomal protease tripeptidyl peptidase 1. LINCL is characterized by lysosomal accumulation of storage material of which only a single protein component, subunit c of mitochondrial ATP synthase, has been well established to date. Identification of other protein constituents of the storage material could provide useful insights into the pathophysiology of disease and the natural substrates for TPP1. We have therefore initiated a proteomic analysis of storage material in brain from a LINCL mouse model. One protein, GFAP (glial fibrillary acidic protein), was found to be elevated in the LINCL mice compared with normal controls in both isolated storage bodies and a lysosome-enriched subcellular fraction that contains storage material. To determine whether GFAP accumulates within the lysosome in LINCL, we examined its intracellular distribution using subcellular fractionation and morphological methods. These experiments demonstrate that GFAP is not a component of the storage material in LINCL, suggesting that reports of GFAP storage in other NCLs may need to be re-examined. A number of other proteins were elevated in the storage material and/or lysosome-enriched fraction from the LINCL mice, but it remains unclear whether these proteins are true constituents of the storage material or, like GFAP, whether they associate with this material upon purification.

Background Information: mtDNA (mitochondrial DNA) mutations that impair oxidative phosphorylation can contribute to carcinogenesis through the increased production of reactive oxygen species and through the release of proteins involved in cell motility and invasion. On the other hand, many human cancers are associated with both the up-regulation and the increased secretion of several proteases and heparanase. In the present study, we tried to determine whether the depletion in mtDNA could modulate the expression and/or the secretion of some lysosomal hydrolases in the 143B osteosarcoma cells, as these mtDNA-depleted cells are characterized by a higher degree of invasiveness than the parental cells., Results: In comparison with the parental cells, we measured a higher amount of procathepsin B in the conditioned culture medium of the 143B cells lacking mtDNA (rho(0) 143B cells), as well as a rise in the specific activity of intracellular cathepsin B. In addition, we observed an activation of the transcription factor NF-kappaB (nuclear factor kappaB) in the cells devoid of functional mitochondria. Finally, we demonstrated that the down-regulation of the NF-kappaB p65 subunit by RNA interference led to a reduction in cathepsin B expression in rho(0) 143B cells., Conclusions: The up-regulation of cathepsin B by NF-kappaB, followed by its secretion into the extracellular environment, might be partly responsible for the previously reported invasiveness of the mtDNA-depleted 143B osteosarcoma cells.

Most newly synthesized proteins destined for the lysosome reach this location via a specific intracellular pathway. In the Golgi, a phosphotransferase specifically labels lysosomal proteins with mannose 6-phosphate (Man-6-P). This modification is recognized by receptors that target the lysosomal proteins to the lysosome where, in most cell types, the Man-6-P recognition marker is rapidly removed. Despite extensive characterization of this pathway, the enzyme responsible for the removal of the targeting modification has remained elusive. In this study, we have identified this activity. Preliminary investigations using a cell-based bioassay were used to follow a dephosphorylation activity that was associated with the lysosomal fraction. This activity was high in the liver, where endogenous lysosomal proteins are efficiently dephosphorylated, but present at a much lower level in the brain, where the modification persists. This observation, combined with an analysis of the expression of lysosomal proteins in different tissues, led us to identify acid phosphatase 5 (ACP5) as a candidate for the enzyme that removes Man-6-P. Expression of ACP5 in N1E-115 neuroblastoma cells, which do not efficiently dephosphorylate lysosomal proteins, significantly decreased the steady state levels of Man6-P glycoproteins. Analysis of ACP5-deficient mice revealed that levels of Man-6-P glycoproteins were highly elevated in tissues that normally express ACP5, and this resulted from a failure to dephosphorylate lysosomal proteins. These results indicate a central role for ACP5 in removal of the Man-6-P recognition marker and open up new avenues to investigate the importance of this process in cell biology and medicine.

Transport of newly synthesized lysosomal membrane proteins from the TGN (trans-Golgi network) to the lysosomes is due to the presence of specific signals in their cytoplasmic domains that are recognized by cytosolic adaptors. p40, a hypothetical transporter of 372 amino acids localized in the lysosomal membrane, contains four putative lysosomal sorting motifs in its sequence: three of the YXXphi-type (Y(6)QLF, Y(106)VAL, Y(333)NGL) and one of the [D/E]XXXL[L/I]-type (EQERL(360)L(361)). To test the role of these motifs in the biosynthetic transport of p40, we replaced the most critical residues of these consensus sequences, the tyrosine residue or the leucine-leucine pair, by alanine or alanine-valine respectively. We analysed the subcellular localization of the mutated p40 proteins in transfected HeLa cells by confocal microscopy and by biochemical approaches (subcellular fractionation on self-forming Percoll density gradients and cell surface biotinylation). The results of the present study show that p40 is mistargeted to the plasma membrane when its dileucine motif is disrupted. No role of the tyrosine motifs could be put forward. Taken together, our results provide evidence that the sorting of p40 from the TGN to the lysosomes is directed by the dileucine EQERL(360)L(361) motif situated in its C-terminal tail.