The LAMP-like protein p67 plays an essential role in the lysosome of African trypanosomes

RNAi knockdown was employed to study the function of p67, a lysosome-associated membrane protein (LAMP)-like type I transmembrane lysosomal glycoprotein in African trypanosomes. Conditional induction of p67 dsRNA resulted in specific approximately 90% reductions in de novo p67 synthesis in both mammalian bloodstream and procyclic insect-stage parasites. Bloodstream cell growth was severely retarded with extensive death after > 24 h of induction. Biosynthetic trafficking of residual p67, and of the soluble lysosomal protease trypanopain, were unimpaired. Endocytosis of tomato lectin, a surrogate receptor-mediated cargo, was only mildly impaired (approximately 20%), but proper lysosomal targeting was unaffected. p67 ablation had dramatic effects on lysosomal morphology with gross enlargement (four- to fivefold) and internal membrane profiles reminiscent of autophagic vacuoles. Ablation of p67 expression rendered bloodstream trypanosomes refractory to lysis by human trypanolytic factor (TLF), a lysosomally activated host innate immune mediator. Similar effects on lysosomal morphology and TLF sensitivity were also obtained by two pharmacological agents that neutralize lysosomal pH--chloroquine and bafilomycin A1. Surprisingly, however, lysosomal pH was not affected in ablated cells suggesting that other physiological alterations must account for increased resistance to TLF. These results indicate p67 plays an essential role in maintenance of normal lysosomal structure and physiology in bloodstream-stage African trypanosomes.     

A dileucine motif and a cluster of acidic amino acids in the second cytoplasmic domain of the batten disease-related CLN3 protein are required for efficient lysosomal targeting

The juvenile form of ceroid lipofuscinosis (Batten disease) is a neurodegenerative lysosomal storage disorder caused by mutations in the CLN3 gene. CLN3 encodes a multimembrane-spanning protein of unknown function, which is mainly localized in lysosomes in non-neuronal cells and in endosomes in neuronal cells. For this study we constructed chimeric proteins of three CLN3 cytoplasmic domains fused to the lumenal and transmembrane domains of the reporter proteins LAMP-1 and lysosomal acid phosphatase to identify lysosomal targeting motifs and to determine the intracellular transport and subcellular localization of the chimera in transfected cell lines. We report that a novel type of dileucine-based sorting motif, EEEX(8)LI, present in the second cytoplasmic domain of CLN3, is sufficient for proper targeting to lysosomes. The first cytoplasmic domain of CLN3 and the mutation of the dileucine motif resulted in a partial missorting of chimeric proteins to the plasma membrane. At equilibrium, 4-13% of the different chimera are present at the cell surface. Analysis of lysosome-specific proteolytic processing revealed that lysosomal acid phosphatase chimera containing the second cytoplasmic domain of CLN3 showed the highest rate of lysosomal delivery, whereas the C terminus of CLN3 was found to be less efficient in lysosomal targeting. However, none of these cytosolic CLN3 domains was able to interact with AP-1, AP-3, or GGA3 adaptor complexes. These data revealed that lysosomal sorting motifs located in an intramolecular cytoplasmic domain of a multimembrane-spanning protein have different structural requirements for adaptor binding than sorting signals found in the C-terminal cytoplasmic domains of single- or dual-spanning lysosomal membrane proteins.     

Lysosomal Targeting of the CLN7 Membrane Glycoprotein and Transport Via the Plasma Membrane Require a Dileucine Motif

CLN7 is a polytopic lysosomal membrane protein deficient in variant late infantile neuronal ceroid lipofuscinosis, a neurodegenerative lysosomal storage disorder. In this study fluorescence protease protection assays and mutational analyses revealed the N‐ and C‐terminal tails of CLN7 in the cytosol and two N‐glycosylation sites at N371 and N376. Both partially and non‐glycosylated CLN7 were correctly transported to lysosomes. To identify lysosomal targeting motifs, we generated CD4‐chimera fused to the N‐ and C‐terminal domains of CLN7. Lysosomal localization of the chimeric proteins requires a consensus acidic dileucine‐based motif in the N‐terminus and two tandem tyrosine‐based signals in the C‐terminus. Mutation of these sorting motifs resulted in cell surface redistribution of CD4 chimeras. However, the dileucine‐based motif is of critical importance for lysosomal localization of the full‐length CLN7 in different cell lines. Cell surface biotinylation revealed that at equilibrium 22% of total CLN7 is localized at the plasma membrane. Mutation of the dileucine motif or the co‐expression of dominant‐negative mutant dynamin K44A led to a further increase of CLN7 at the plasma membrane. Our data demonstrate that CLN7 contains several cytoplasmic lysosomal targeting signals of which the N‐terminal dileucine‐based motif is required for the predominant lysosomal targeting along the indirect pathway and clathrin‐mediated endocytosis of CLN7.     

Targeting of NPC1 to late endosomes involves multiple signals, including one residing within the putative sterol-sensing domain

The NPC1 protein is a multipass transmembrane protein whose deficiency causes the autosomal recessive lipid storage disorder Niemann-Pick type C1. NPC1 localizes predominantly to late endosomes and has a dileucine motif located within a small cytoplasmic tail thought to target the protein to this location. Our data have suggested previously that the protein can reach its correct location in the absence of its cytoplasmic tail, suggesting that other signals contribute to NPC1 targeting. By using various FLAG-tagged and CD32-NPC1 chimeric fusion constructs, we show that multiple signals are responsible for the trafficking of NPC1 to the endosomal compartment, including the dileucine motif and a previously unidentified signal residing within the putative sterol-sensing domain transmembrane domain 3. Neither region alone was capable of directing heterologous CD32 fusions to late endosomes exclusively via the trans-Golgi network to the late endosome route taken by wild-type NPC1; transmembrane domain 3 was unable to maintain CD32 in late endosomes, indicating that two or more signals work in concert to target and retain NPC1 in this compartment. In addition we confirm that the tail dileucine motif is not essential for NPC1 targeting to late endosomes, and we discuss the implications of this finding along with the previously unappreciated role for transmembrane domain 3 in NPC1 localization and function.     

Role of AP-1 in Developmentally Regulated Lysosomal Trafficking in Trypanosoma brucei

African trypanosomes are the causative agents of human trypanosomiasis (sleeping sickness). The pathogenic stage of the parasite has unique adaptations to life in the bloodstream of the mammalian host, including upregulation of endocytic and lysosomal activities. We investigated stage-specific requirements for cytoplasmic adaptor/clathrin machinery in post-Golgi apparatus biosynthetic sorting to the lysosome using RNA interference silencing of the Tbμ1 subunit of adaptor complex 1 (AP-1), in conjunction with immunolocalization, kinetic analyses of reporter transport, and quantitative endocytosis assays. Tbμ1 silencing was lethal in both stages, indicating a critical function(s) for the AP-1 machinery. Transport of soluble and membrane-bound secretory cargoes was Tbμ1 independent in both stages. In procyclic parasites, trafficking of the lysosomal membrane protein, p67, was disrupted, leading to cell surface mislocalization. The lysosomal protease trypanopain was also secreted, suggesting a transmembrane-sorting receptor for this soluble hydrolase. In bloodstream trypanosomes, both p67 and trypanopain trafficking were unaffected by Tbμ1 silencing, suggesting that AP-1 is not necessary for biosynthetic lysosomal trafficking. Endocytosis in bloodstream cells was also unaffected, indicating that AP-1 does not function at the flagellar pocket. These results indicate that post-Golgi apparatus sorting to the lysosome is critically dependent on the AP-1/clathrin machinery in procyclic trypanosomes but that this machinery is not necessary in bloodstream parasites. We propose a simple model for stage-specific default secretory trafficking in trypanosomes that is consistent with the behavior of other soluble and glycosylphosphatidylinositol-anchored cargos and which is influenced by upregulation of endocytosis in bloodstream parasites as an adaptation to life in the mammalian bloodstream.African trypanosomes (Trypanosoma brucei subspecies), the agents of African sleeping sickness, are alone among the kinetoplastid parasites (including Trypanosoma cruzi and Leishmania spp.) in having a pathogenic bloodstream stage that exists and replicates extracellularly in the mammalian host. This places unique constraints on the parasite in terms of dealing with host immune responses and on acquisition of essential nutrients. The parasite has evolved many strategies to deal with these constraints, the best known of which is the process of antigenic variation (9). Another is the lysosome, which impacts the host-pathogen balance in multiple ways. Trypanosomes have a single terminal lysosome that is the final repository of endocytic cargo acquired from the host serum for nutritional purposes (30), as well as for potentially lytic immune complexes removed from the cell surface (4, 8). Both endocytosis and lysosomal hydrolytic activities are differentially regulated through the trypanosome life cycle (11, 30), and there are stage-specific differences in the biosynthetic trafficking of essential lysosomal components (discussed below). The release of lysosomal proteases is a factor in the signature event of human infection, penetration of the central nervous system (36). Finally, lysosomal physiology is critical to the activity of an innate human serum resistance trait, trypanolytic factor, which limits the host range of Trypanosoma species (38).Clearly, given its multiple roles in pathogenesis, biogenesis of the lysosome is critical to the success of trypanosomes as human parasites. As in all eukaryotes, lysosomal biogenesis is a balance between the proper sorting of newly synthesized membranes and proteins and recycling of established membranes and proteins internalized from the cell surface. In each case, protein sorting involves recognition of specific signals in cargo molecules by cellular machinery for inclusion in nascent transport vesicles destined for downstream delivery. Unique sets of cytoplasmic coat complexes at discrete intracellular locations serve the dual purpose of simultaneously mediating vesicle formation and selective cargo loading. The best characterized of these machineries is the clathrin/adaptin system for formation of coated vesicles at the Golgi apparatus and the plasma membrane (10, 41). Adaptor complexes (APs) are cytosolic heterotetramers that interact with specific signals in the cytoplasmic domains of membrane cargo proteins, such as dileucine motifs ([E/D]XXXL[L/I]) and tyrosine motifs (YXXØ, where Ø is a bulky hydrophobic residue). The prototypic AP complexes are AP-1 and AP-2, which function at the trans-Golgi network and plasma membrane, respectively. Both are composed of two large subunits (γ/β1 in AP-1; α/β2 in AP-2) and two smaller subunits (σ1/μ1 in AP-1; σ2/μ2 in AP-2). YXXØ motifs interact with μ adaptins, and dileucine motifs interact with combinations of adaptin subunits in both AP-1 and AP-2 (26, 40, 42). It is the large subunits, particularly β adaptin, that mediate clathrin recruitment (19, 44). Other APs, AP-3 and AP-4, with discrete subunit compositions, also exist. AP-3 functions in trafficking to lysosome-related organelles, such as melanosomes, and AP-4 may be involved in basolateral trafficking in polarized epithelial cells (10). The genome of the African trypanosome, T. brucei, encodes a complete complement of orthologous subunits for AP-1, AP-3, and AP-4 but has no genes for AP-2, the major adaptor complex mediating endocytosis in vertebrate cells (16). This is likely due to evolutionary loss, since the closely related T. cruzi has orthologues of all four APs.Two major lysosomal cargo proteins have been studied in T. brucei, the LAMP (lysosome-associated membrane protein)-like protein p67 and the cathepsin L orthologue trypanopain. p67 is a type I membrane protein with a large glycosylated lumenal domain and a short cytoplasmic domain (1, 27). In procyclic insect stage (PCF) trypanosomes, the cytoplasmic domain is both necessary and sufficient for lysosomal targeting of a heterologous reporter, and its deletion results in mistargeting of p67 to the cell surface (1). The cytoplasmic domain contains two canonical dileucine motifs, mutation of which also results in delivery to the cell surface (47). These findings strongly indicate the existence of cognate cytoplasmic machinery for lysosomal delivery of p67 in PCF trypanosomes. Strikingly, however, the cytoplasmic domain, and its motifs, are totally dispensable for lysosomal targeting in bloodstream stage (BSF) trypanosomes (1). Deletion of the cytoplasmic domain results in minor mislocalization to the cell surface, but p67 is still overwhelmingly delivered to the lysosome. Ongoing lysosomal targeting cannot easily be attributed to misfolding of the lumenal domain, as suggested by others (3), since the normal transport-associated patterns of p67 glycosylation and cleavage prevail in these deletion constructs.Less is known about targeting of soluble trypanopain. In mammalian cells, soluble hydrolases are targeted to the lysosome by the addition of mannose-6-phosphate (M6P) moieties in the Golgi apparatus, which serve as ligands for recognition and lysosomal targeting by downstream M6P receptors (28). Soluble hydrolases can also be sorted by receptors that recognize polypeptide motifs, such as sortilins in mammalian cells (12) and Vps10 in yeast (13, 32). These receptors have lumenal cargo recognition domains and cytoplasmic domains containing signals for late endosomal targeting and recycling. M6P-modified N-linked glycans are not found in trypanosomes, and genes encoding the necessary enzymatic activities are absent from the genome (16), ruling out this possibility for trypanopain sorting. However, the T. cruzi orthologue, cruzipain, has been shown to rely on peptide motifs in the N-terminal prodomain for targeting (24), raising the possibility of a sortilin/Vps10p-like sorting receptor. Although there are no obvious orthologues of these proteins in the T. brucei genome, overexpression of trypanopain in PCF trypanosomes leads to secretion, an observation that is consistent with saturation of a specific sorting receptor (S. S. Sutterwala and J. D. Bangs, unpublished observations).Having previously studied the innate signals involved in p67 targeting (1, 47), we now turned our attention to the cognate machinery for post-Golgi apparatus sorting. Specifically, we investigate the role of trypanosomal AP-1 in stage-specific biosynthetic trafficking to the lysosome using RNA interference (RNAi)-mediated silencing of the Tbμ1 (geneDB no. Tb927.7.3180 [www.genedb.org]) subunit as our primary strategy. Our results demonstrate that AP-1 and clathrin are critical for lysosomal targeting of p67 and trypanopain in PCF trypanosomes but that they are essentially dispensable in BSF parasites. These data, in conjunction with the behavior of p67-targeting mutants (1) and other trypanosomal secretory reporters, lead us to propose a simple model for stage-specific default trafficking in African trypanosomes. Although in some respects our results are similar to those of a recent publication using RNAi silencing of the Tbγ1 subunit of AP-1 (3), they differ in key aspects, leading us to significantly different conclusions.     

AP-1 and AP-3 facilitate lysosomal targeting of Batten disease protein CLN3 via its dileucine motif

CLN3 is a transmembrane protein with a predominant localization in lysosomes in non-neuronal cells but is also found in endosomes and the synaptic region in neuronal cells. Mutations in the CLN3 gene result in juvenile neuronal ceroid lipofuscinosis or Batten disease, which currently is the most common cause of childhood dementia. We have recently reported that the lysosomal targeting of CLN3 is facilitated by two targeting motifs: a dileucine-type motif in a cytoplasmic loop domain and an unusual motif in the carboxyl-terminal cytoplasmic tail comprising a methionine and a glycine separated by nine amino acids (Kyttala, A., Ihrke, G., Vesa, J., Schell, M. J., and Luzio, J. P. (2004) Mol. Biol. Cell 15, 1313-1323). In the present study, we investigated the pathways and mechanisms of CLN3 sorting using biochemical binding assays and immunofluorescence methods. The dileucine motif of CLN3 bound both AP-1 and AP-3 in vitro, and expression of mutated CLN3 in AP-1- or AP-3-deficient mouse fibroblasts showed that both adaptor complexes are required for sequential sorting of CLN3 via this motif. Our data indicate the involvement of complex sorting machinery in the trafficking of CLN3 and emphasize the diversity of parallel and sequential sorting pathways in the trafficking of membrane proteins.     

The amino acids upstream of NH(2)-terminal dileucine motif play a role in regulating the intracellular sorting of the Class III transporters GLUT8 and GLUT12

Abstract

The transport of glucose across cell membranes is mediated by a family of facilitative glucose transporters (GLUTs). The class III glucose transporters GLUT8 and GLUT12 both contain a similar [DE]XXXL[LI] dileucine sorting signal in their amino terminus. This type of dileucine motif facilitates protein trafficking to various organelles or to the plasma membrane via interactions with adaptor protein (AP) complexes. The [DE]XXXL[LI] motif in GLUT8 is thought to direct it to late endosomal/lysosomal compartments via its interactions with AP1 and AP2. Unlike GLUT8, the [DE]XXXL[LI] motif does not direct GLUT12 to a lysosomal compartment. Rather, GLUT12 resides in the Golgi network and at the plasma membrane. In a previous study, we found that exchanging the XXX (TQP) residues in GLUT8 with the corresponding residues in GLUT12 (GPN) resulted in a dramatic missorting of GLUT8 to the cell surface. We postulated that the XXX amino acids upstream of the dileucine motif in GLUT8 influence the degree of interaction between the [DE]XXXL[LI] motif and adaptor proteins. To further explore its trafficking mechanisms, we created mutant constructs to identify the role that each of the individual XXX amino acids has for regulating the intracellular sorting of GLUT8. Here we find that the XXX amino acids, specifically the position of a proline -2 from the dileucine residues, influence the affinity of APs for GLUT8 and GLUT12.     

The amino acids upstream of NH(2)-terminal dileucine motif play a role in regulating the intracellular sorting of the Class III transporters GLUT8 and GLUT12

The transport of glucose across cell membranes is mediated by a family of facilitative glucose transporters (GLUTs). The class III glucose transporters GLUT8 and GLUT12 both contain a similar [DE]XXXL[LI] dileucine sorting signal in their amino terminus. This type of dileucine motif facilitates protein trafficking to various organelles or to the plasma membrane via interactions with adaptor protein (AP) complexes. The [DE]XXXL[LI] motif in GLUT8 is thought to direct it to late endosomal/lysosomal compartments via its interactions with AP1 and AP2. Unlike GLUT8, the [DE]XXXL[LI] motif does not direct GLUT12 to a lysosomal compartment. Rather, GLUT12 resides in the Golgi network and at the plasma membrane. In a previous study, we found that exchanging the XXX (TQP) residues in GLUT8 with the corresponding residues in GLUT12 (GPN) resulted in a dramatic missorting of GLUT8 to the cell surface. We postulated that the XXX amino acids upstream of the dileucine motif in GLUT8 influence the degree of interaction between the [DE]XXXL[LI] motif and adaptor proteins. To further explore its trafficking mechanisms, we created mutant constructs to identify the role that each of the individual XXX amino acids has for regulating the intracellular sorting of GLUT8. Here we find that the XXX amino acids, specifically the position of a proline -2 from the dileucine residues, influence the affinity of APs for GLUT8 and GLUT12.     

Late endosomal Rab7 regulates lysosomal trafficking of endocytic but not biosynthetic cargo in Trypanosoma brucei

We present the first functional analysis of the small GTPase, TbRab7, in Trypanosoma brucei. TbRab7 defines discrete late endosomes closely juxtaposed to the terminal p67(+) lysosome. RNAi indicates that TbRab7 is essential in bloodstream trypanosomes. Initial rates of endocytosis were unaffected, but lysosomal delivery of cargo, including tomato lectin (TL) and trypanolytic factor (TLF) were blocked. These accumulate in a dispersed internal compartment of elevated pH, likely derived from the late endosome. Surface binding of TL but not TLF was reduced, suggesting that cellular distribution of flagellar pocket receptors is differentially regulated by TbRab7. TLF activity was reduced approximately threefold confirming that lysosomal delivery is critical for trypanotoxicity. Unexpectedly, delivery of endogenous proteins, p67 and TbCatL, were unaffected indicating that TbRab7 does not regulate biosynthetic lysosomal trafficking. Thus, unlike mammalian cells and yeast, lysosomal trafficking of endocytosed and endogenous proteins occur via different routes and/or are regulated differentially. TbRab7 silencing had no effect on a cryptic default pathway to the lysosome, suggesting that the default lysosomal reporters p67ΔTM, p67ΔCD and VSGΔGPI do not utilize the endocytic pathway as previously proposed. Surprisingly, conditional knockout indicates that TbRab7 may be non-essential in procyclic insect form trypanosomes.     

YXXM motifs in the PDGF-beta receptor serve dual roles as phosphoinositide 3-kinase binding motifs and tyrosine-based endocytic sorting signals

Phosphoinositide 3-kinases (PI 3-kinases) are important regulators of endocytic trafficking. Previous studies have shown that mutant human platelet-derived growth factor-beta receptors (PDGFR), which contain Phe in place of Tyr at the two p85/p110 PI 3-kinase binding sites (PDGFR-F/F), are defective for both p85 binding and ligand-stimulated degradation. This suggested that p85/p110 regulates PDGFR trafficking. However, more recent work has identified hVPS34, and not p85/p110, as the major PI 3-kinase regulating the movement of receptors through the endosomal system. To reconcile this discrepancy, we hypothesized that YXXM motifs in the PDGFR might play a second role as Tyr-based lysosomal sorting motifs (YXXPhi). To test this, we replaced both YXXM motifs with a motif from LAMP-1, YQTI. This mutant PDGFR (PDGFR-YQTI) still underwent PDGF-stimulated autophosphorylation but did not bind p85. In CHO cells, both wild-type and YQTI receptors showed PDGF-stimulated turnover, whereas F/F receptors did not. In addition, uptake and degradation of cell surface-labeled YXXM and YQTI receptors was fast relative to F/F receptors. We also constructed chimeras containing extracellular and membrane-spanning domains from CD25 (Tac) and cytoplasmic tails containing the YQTI motif, two YXXM motifs, or two mutant FXXM motifs. The YXXM and YQTI chimeras mediated lysosomal delivery of fluorescein isothiocyanate-labeled anti-CD25 antibodies, whereas the F/F chimera was defective. Thus, YQTI motifs can target PDGFR for degradation in the absence of p85/p110 binding, and the p85/p110 binding motifs from PDGFR are sufficient to target Tac chimeras to the lysosome. These data suggest that the YXXM motifs in the PDGFR serve two distinct functions: PI 3-kinase recruitment and lysosomal targeting.     

Functional characterization of a lysosomal sorting motif in the cytoplasmic tail of HLA-DObeta

HLA-DO is an intracellular non-classical class II major histocompatibility complex molecule expressed in the endocytic pathway of B lymphocytes, which regulates the loading of antigenic peptides onto classical class II molecules such as HLA-DR. The activity of HLA-DO is mediated through its interaction with the peptide editor HLA-DM. Here, our results demonstrate that although HLA-DO is absolutely dependent on its association with DM to egress the endoplasmic reticulum, the cytoplasmic portion of its beta chain encodes a functional lysosomal sorting signal. By confocal microscopy and flow cytometry analysis, we show that reporter transmembrane molecules fused to the cytoplasmic tail of HLA-DObeta accumulated in Lamp-1(+) vesicles of transfected HeLa cells. Mutagenesis of a leucine-leucine motif abrogated lysosomal accumulation and resulted in cell surface redistribution of reporter molecules. Finally, we show that mutation of the di-leucine sequence in DObeta did not alter its lysosomal sorting when associated with DM molecules. Taken together, these results demonstrate that lysosomal expression of the DO-DM complex is mediated primarily by the tyrosine-based motif of HLA-DM and suggest that the DObeta-encoded motif is involved in the fine-tuning of the intracellular sorting.     

A dileucine motif targets E-cadherin to the basolateral cell surface in Madin-Darby canine kidney and LLC-PK1 epithelial cells

E-cadherin is a major adherens junction protein of epithelial cells, with a central role in cell-cell adhesion and cell polarity. Newly synthesized E-cadherin is targeted to the basolateral cell surface. We analyzed targeting information in the cytoplasmic tail of E-cadherin by utilizing chimeras of E-cadherin fused to the ectodomain of the interleukin-2alpha (IL-2alpha) receptor expressed in Madin-Darby canine kidney and LLC-PK(1) epithelial cells. Chimeras containing the full-length or membrane-proximal half of the E-cadherin cytoplasmic tail were correctly targeted to the basolateral domain. Sequence analysis of the membrane-proximal tail region revealed the presence of a highly conserved dileucine motif, which was analyzed as a putative targeting signal by mutagenesis. Elimination of this motif resulted in the loss of Tac/E-cadherin basolateral localization, pinpointing this dileucine signal as being both necessary and sufficient for basolateral targeting of E-cadherin. Truncation mutants unable to bind beta-catenin were correctly targeted, showing, contrary to current understanding, that beta-catenin is not required for basolateral trafficking. Our results also provide evidence that dileucine-mediated targeting is maintained in LLC-PK(1) cells despite the altered polarity of basolateral proteins with tyrosine-based signals in this cell line. These results provide the first direct insights into how E-cadherin is targeted to the basolateral membrane.     

Two motifs target Batten disease protein CLN3 to lysosomes in transfected nonneuronal and neuronal cells

Batten disease is a neurodegenerative disorder resulting from mutations in CLN3, a polytopic membrane protein, whose predominant intracellular destination in nonneuronal cells is the lysosome. The topology of CLN3 protein, its lysosomal targeting mechanism, and the development of Batten disease are poorly understood. We provide experimental evidence that both the N and C termini and one large loop domain of CLN3 face the cytoplasm. We have identified two lysosomal targeting motifs that mediate the sorting of CLN3 in transfected nonneuronal and neuronal cells: an unconventional motif in the long C-terminal cytosolic tail consisting of a methionine and a glycine separated by nine amino acids [M(X)9G], and a more conventional dileucine motif, located in the large cytosolic loop domain and preceded by an acidic patch. Each motif on its own was sufficient to mediate lysosomal targeting, but optimal efficiency required both. Interestingly, in primary neurons, CLN3 was prominently seen both in lysosomes in the cell body and in endosomes, containing early endosomal antigen-1 along neuronal processes. Because there are few lysosomes in axons and peripheral parts of dendrites, the presence of CLN3 in endosomes of neurons may be functionally important. Endosomal association of the protein was independent of the two lysosomal targeting motifs.     

Two di-leucine motifs regulate trafficking of mucolipin-1 to lysosomes

Mutations in the mucolipin-1 gene have been linked to mucolipidosis type IV, a lysosomal storage disorder characterized by severe neurological and ophthalmologic abnormalities. Mucolipin-1 is a membrane protein containing six putative transmembrane domains with both its N- and C-termini localized facing the cytosol. To gain information on the sorting motifs that mediate the trafficking of this protein to lysosomes, we have generated chimeras in which the N- and C- terminal tail portions of mucolipin-1 were fused to a reporter gene. In this article, we report the identification of two separate di-leucine-type motifs that co-operate to regulate the transport of mucolipin-1 to lysosomes. One di-leucine motif is positioned at the N-terminal cytosolic tail and mediates direct transport to lysosomes, whereas the other di-leucine motif is found at the C-terminal tail and functions as an adaptor protein 2-dependent internalization motif. We have also found that the C-terminal tail of mucolipin-1 is palmitoylated and that this modification might regulate the efficiency of endocytosis. Finally, the mutagenesis of both di-leucine motifs abrogated lysosomal accumulation and resulted in cell-surface redistribution of mucolipin-1. Taken together, these results reveal novel information regarding the motifs that regulate mucolipin-1 trafficking and suggest a role for palmitoylation in protein sorting.     

Topology and endoplasmic reticulum retention signals of the lysosomal storage disease-related membrane protein CLN6

CLN6 is a polytopic membrane protein of unknown function resident in the endoplasmic reticulum (ER). Mutant CLN6 causes the lysosomal storage disorder neuronal ceroid lipofuscinosis. Defining the topology of CLN6, and the structural domains and motifs required for interaction with cytosolic and luminal proteins may allow insights into its function. In this study we analysed the topology, ER retention and oligomerization of CLN6. We demonstrated, by differential membrane permeabilization of transfected BHK cells using specific detergents and two distinct antibodies, that CLN6 contains an N-terminal cytoplasmic domain, seven transmembrane domains, and a luminal C terminus. Mutational analyses and confocal immunofluorescence microscopy showed that changes of potential ER localization signals in the N- or C-terminal domain (a triple arginine cluster, and a dileucine motif) did not alter the subcellular localization of CLN6. The deletion of a dilysine motif impaired partially the ER localization of CLN6. Furthermore, expression analyses of fusion and deletion constructs in non-neuronal and neuronal cells suggested that two portions of CLN6 contributed to its retention within the ER. We showed that the N-terminal domain was necessary but not sufficient for ER retention of CLN6 and that deletion of transmembrane domains 6 and 7 was accompanied with the loss of ER localization and, in some instances, trafficking to the cisGolgi. From these data we concluded that CLN6 maintains its ER localization by expressing retention signals present in both the N-terminal cytosolic domain and in the carboxy-proximal transmembrane domains 6 and 7. Additionally, the ability of CLN6 to homodimerize may also prevent exit from the ER via an interaction with membrane-associated factors.     

Topology and endoplasmic reticulum retention signals of the lysosomal storage disease-related membrane protein CLN6

CLN6 is a polytopic membrane protein of unknown function resident in the endoplasmic reticulum (ER). Mutant CLN6 causes the lysosomal storage disorder neuronal ceroid lipofuscinosis. Defining the topology of CLN6, and the structural domains and motifs required for interaction with cytosolic and luminal proteins may allow insights into its function. In this study we analysed the topology, ER retention and oligomerization of CLN6. We demonstrated, by differential membrane permeabilization of transfected BHK cells using specific detergents and two distinct antibodies, that CLN6 contains an N-terminal cytoplasmic domain, seven transmembrane domains, and a luminal C terminus. Mutational analyses and confocal immunofluorescence microscopy showed that changes of potential ER localization signals in the N- or C-terminal domain (a triple arginine cluster, and a dileucine motif) did not alter the subcellular localization of CLN6. The deletion of a dilysine motif impaired partially the ER localization of CLN6. Furthermore, expression analyses of fusion and deletion constructs in non-neuronal and neuronal cells suggested that two portions of CLN6 contributed to its retention within the ER. We showed that the N-terminal domain was necessary but not sufficient for ER retention of CLN6 and that deletion of transmembrane domains 6 and 7 was accompanied with the loss of ER localization and, in some instances, trafficking to the cisGolgi. From these data we concluded that CLN6 maintains its ER localization by expressing retention signals present in both the N-terminal cytosolic domain and in the carboxy-proximal transmembrane domains 6 and 7. Additionally, the ability of CLN6 to homodimerize may also prevent exit from the ER via an interaction with membrane-associated factors.     

Study of the mouse sortilin gene: Effects of its transient silencing by RNA interference in TM4 Sertoli cells

Using databases of the mouse genome in combination with a sequence deduced from a mouse sortilin cDNA originated in our laboratory, we found the sortilin gene to map to a region of chromosome 3. The mouse sortilin gene contains 19 short exons separated by introns of various sizes. The study elucidated the exon-intron boundaries. Some introns extend over more than 24 kb. In the cytoplasmic domain of the translation product, we found a dileucine motif and three other motifs known to constitute the active sorting signal of the mannose 6-phosphate receptor (M6P-R). We also tested the hypothesis that sortilin is involved in the sorting of prosaposin (SGP-1) to the lysosomes. Prosaposin was initially identified in Sertoli cells, found in large amounts in the lysosomal compartment and implicated in the degradation of residual bodies released by the spermatids during spermiation. Interestingly, the targeting of prosaposin to the lysosomes is independent of the M6P-R. This investigation demonstrated that sortilin was required for the trafficking of prosaposin to the lysosomes in TM4 cells. The requirement of sortilin was shown using a siRNA probe to block the translation of sortilin mRNA. Sortilin-deficient cells were not able to route prosaposin to the lysosomal compartment but continue to transport cathepsin B, since this hydrolase uses the M6P-R to be routed to the lysosomes. These results indicate that sortilin appears to be involved in the lysosomal trafficking of prosaposin.     

Lysosomal localization of murine CD1d mediated by AP-3 is necessary for NK T cell development

The presentation of lipid and glycolipid Ags to T cells is mediated through CD1 molecules. In the mouse and rat only a single isoform, CD1d, performs these functions, while humans and all other mammals studied have members of both group I (CD1a, -b, and -c) and group II (CD1d) isoforms. Murine CD1d contains a cytoplasmic tyrosine-based sorting motif that is similar to motifs recognized by adaptor protein complexes that sort transmembrane proteins. Here we show that the adaptor protein complex, AP-3, directly interacts with murine CD1d and controls its targeting to lysosomes. AP-3 deficiency results in a redistribution of CD1d from lysosomes to the cell surface of thymocytes, B cell-depleted splenocytes, and dendritic cells. The altered trafficking of CD1d in AP-3-deficient mice results in a significant reduction of NK1.1(+)TCR-beta(+) and CD1d tetramer-positive cells, consistent with a defect in CD1d self-Ag presentation and thymocyte-positive selection. The AP-3 complex has recently been shown to associate with the human CD1b isoform, which has an intracellular distribution pattern similar to that of murine CD1d. We propose that lysosomal sampling may be so critical for efficient host defense that mice have evolved mechanisms to target their single CD1 isoform to lysosomes for sampling lipid Ags. Here we show the dominant mechanism for this trafficking is mediated by AP-3.     

Two dileucine motifs mediate late endosomal/lysosomal targeting of transmembrane protein 192 (TMEM192) and a C-terminal cysteine residue is responsible for disulfide bond formation in TMEM192 homodimers

TMEM192 (transmembrane protein 192) is a novel constituent of late endosomal/lysosomal membranes with four potential transmembrane segments and an unknown function that was initially discovered by organellar proteomics. Subsequently, localization in late endosomes/lysosomes has been confirmed for overexpressed and endogenous TMEM192, and homodimers of TMEM192 linked by disulfide bonds have been reported. In the present study the molecular determinants of TMEM192 mediating its transport to late endosomes/lysosomes were analysed by using CD4 chimaeric constructs and mutagenesis of potential targeting motifs in TMEM192. Two directly adjacent N-terminally located dileucine motifs of the DXXLL-type were found to be critical for transport of TMEM192 to late endosomes/lysosomes. Whereas disruption of both dileucine motifs resulted in mistargeting of TMEM192 to the plasma membrane, each of the two motifs was sufficient to ensure correct targeting of TMEM192. In order to study disulfide bond formation, mutagenesis of cysteine residues was performed. Mutation of Cys266 abolished disulfide bridge formation between TMEM192 molecules, indicating that TMEM192 dimers are linked by a disulfide bridge between their C-terminal tails. According to the predicted topology, Cys266 would be localized in the reductive milieu of the cytosol where disulfide bridges are generally uncommon. Using immunogold labelling and proteinase protection assays, the localization of the N- and C-termini of TMEM192 on the cytosolic side of the late endosomal/lysosomal membrane was experimentally confirmed. These findings may imply close proximity of the C-termini in TMEM192 dimers and a possible involvement of this part of the protein in dimer assembly.     

A conserved dileucine motif mediates clathrin and AP-2-dependent endocytosis of the HIV-1 envelope protein

During the assembly of enveloped viruses viral and cellular components essential for infectious particles must colocalize at specific membrane locations. For the human and simian immunodeficiency viruses (HIV and SIV), sorting of the viral envelope proteins (Env) to assembly sites is directed by trafficking signals located in the cytoplasmic domain of the transmembrane protein gp41 (TM). A membrane proximal conserved GYxx? motif mediates endocytosis through interaction with the clathrin adaptor AP-2. However, experiments with SIV(mac239) Env indicate the presence of additional signals. Here we show that a conserved C-terminal dileucine in HIV(HxB2) also mediates endocytosis. Biochemical and morphological assays demonstrate that the C-terminal dileucine motif mediates internalization as efficiently as the GYxx? motif and that both must be removed to prevent Env internalization. RNAi experiments show that depletion of the clathrin adaptor AP-2 leads to increased plasma membrane expression of HIV Env and that this adaptor is required for efficient internalization mediated by both signals. The redundancy of conserved endocytosis signals and the role of the SIV(mac239) Env GYxx? motif in SIV pathogenesis, suggest that these motifs have functions in addition to endocytosis, possibly related to Env delivery to the site of viral assembly and/or incorporation into budding virions.