Marielle Boonen

Subcellular Trafficking and Activity of Hyal-1 and Its Processed Forms in Murine Macrophages

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 PercollTM 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.

Intracellular localization of p40, a protein identified in a preparation of lysosomal membranes

Unlike lysosomal soluble proteins, few lysosomal membrane proteins have been identified. Rat liver lysosomes were purified by centrifugation on a Nycodenz density gradient. The most hydrophobic proteins were extracted from the lysosome membrane preparation and were identified by MS. We focused our attention on a protein of approx. 40 kDa, p40, which contains seven to ten putative transmembrane domains and four lysosomal consensus sorting motifs in its sequence. Knowing that preparations of lysosomes obtained by centrifugation always contain contaminant membranes, we combined biochemical and morphological methods to analyse the subcellular localization of p40. The results of subcellular fractionation of mouse liver homogenates validate the lysosomal residence of p40. In particular, a density shift of lysosomes induced by Triton WR-1339 similarly affected the distributions of p40 and beta-galactosidase, a lysosomal marker protein. We confirmed by fluorescence microscopy on eukaryotic cells transfected with p40 or p40-GFP (green fluorescent protein) constructs that p40 is localized in lysosomes. A first molecular characterization of p40 in transfected Cos-7 cells revealed that it is an unglycosylated protein tightly associated with membranes. Taken together, our results strongly support the hypothesis that p40 is an authentic lysosomal membrane protein.

Cathepsin D and its newly identified transport receptor SEZ6L2 can modulate neurite outgrowth.

ABSTRACT 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. Highlighted Article: SEZ6L2 participates in the intracellular trafficking of the hydrolase cathepsin D. In turn, proteolytic cleavage of SEZ6L2 by cathepsin D modulates the neurite outgrowth process.

Accounting for Protein Subcellular Localization: A Compartmental Map of the Rat Liver Proteome

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.

Subcellular Trafficking of Mammalian Lysosomal Proteins: An Extended View

Lysosomes clear macromolecules, maintain nutrient and cholesterol homeostasis, participate in tissue repair, and in many other cellular functions. To assume these tasks, lysosomes rely on their large arsenal of acid hydrolases, transmembrane proteins and membrane-associated proteins. It is therefore imperative that, post-synthesis, these proteins are specifically recognized as lysosomal components and are correctly sorted to this organelle through the endosomes. Lysosomal transmembrane proteins contain consensus motifs in their cytosolic regions (tyrosine- or dileucine-based) that serve as sorting signals to the endosomes, whereas most lysosomal acid hydrolases acquire mannose 6-phosphate (Man-6-P) moieties that mediate binding to two membrane receptors with endosomal sorting motifs in their cytosolic tails. These tyrosine- and dileucine-based motifs are tickets for boarding in clathrin-coated carriers that transport their cargo from the trans-Golgi network and plasma membrane to the endosomes. However, increasing evidence points to additional mechanisms participating in the biogenesis of lysosomes. In some cell types, for example, there are alternatives to the Man-6-P receptors for the transport of some acid hydrolases. In addition, several “non-consensus” sorting motifs have been identified, and atypical transport routes to endolysosomes have been brought to light. These “unconventional” or “less known” transport mechanisms are the focus of this review.

Mouse liver lysosomes contain enzymatically active processed forms of Hyal-1

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.

A dileucine signal situated in the C-terminal tail of the lysosomal membrane protein p40 is responsible for its targeting to lysosomes.

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.

SNAT7 is the primary lysosomal glutamine exporter required for extracellular protein-dependent growth of cancer cells

Significance Lysosomes are degradative intracellular organelles essential to cell maintenance and homeostasis. Although their degradative function is well documented, the proteins responsible for the efflux, and reuse, of lysosomal degradation products remain largely unknown. In this study, we identify the transporter responsible for lysosomal efflux of glutamine, an amino acid central to several key metabolic pathways. This central role of glutamine is exploited by several types of cancer cells with increased consumption of glutamine. Interestingly, genetic inactivation of the transporter impairs their growth under conditions of limited glutamine availability when internalized extracellular proteins are used as an alternative source of amino acids, suggesting novel approaches for anticancer therapies. 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.

Monocytes/Macrophages Upregulate the Hyaluronidase HYAL1 and Adapt Its Subcellular Trafficking to Promote Extracellular Residency upon Differentiation into Osteoclasts

Osteoclasts are giant bone-resorbing cells originating from monocytes/macrophages. During their differentiation, they overexpress two lysosomal enzymes, cathepsin K and TRAP, which are secreted into the resorption lacuna, an acidified sealed area in contact with bone matrix where bone degradation takes place. Here we report that the acid hydrolase HYAL1, a hyaluronidase able to degrade the glycosaminoglycans hyaluronic acid (HA) and chondroitin sulfate, is also upregulated upon osteoclastogenesis. The mRNA expression and protein level of HYAL1 are markedly increased in osteoclasts differentiated from RAW264.7 mouse macrophages or primary mouse bone marrow monocytes compared to these precursor cells. As a result, the HYAL1-mediated HA hydrolysis ability of osteoclasts is strongly enhanced. Using subcellular fractionation, we demonstrate that HYAL1 proteins are sorted to the osteoclast lysosomes even though, in contrast to cathepsin K and TRAP, HYAL1 is poorly mannose 6-phosphorylated. We reported previously that macrophages secrete HYAL1 proforms by constitutive secretion, and that these are recaptured by the cell surface mannose receptor, processed in endosomes and sorted to lysosomes. Present work highlights that osteoclasts secrete HYAL1 in two ways, through lysosomal exocytosis and constitutive secretion, and that these cells promote the extracellular residency of HYAL1 through downregulation of the mannose receptor. Interestingly, the expression of the other main hyaluronidase, HYAL2, and of lysosomal exoglycosidases involved in HA degradation, does not increase similarly to HYAL1 upon osteoclastogenesis. Taken together, these findings point out the predominant involvement of HYAL1 in bone HA metabolism and perhaps bone remodeling via the resorption lacuna.

Molecular determinants that mediate the sorting of human ATG9A from the endoplasmic reticulum

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.