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.     

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.     

Protein targeting by tyrosine- and di-leucine-based signals: evidence for distinct saturable components

Targeting of transmembrane proteins to lysosomes, endosomal compartments, or the trans-Golgi network is largely dependent upon cytoplasmically exposed sorting signals. Among the most widely used signals are those that conform to the tyrosine-based motif, YXXO (where Y is tyrosine, X is any amino acid, and O is an amino acid with a bulky hydrophobic group), and to the di-leucine (or LL) motif. Signals conforming to both motifs have been implicated in protein localization to similar post-Golgi compartments. We have exploited the saturability of sorting to ask whether different YXXO or LL signals use shared components of the targeting machinery. Chimeric proteins containing various cytoplasmic domains and/or targeting signals were overexpressed in HeLa cells by transient transfection. Endogenous transferrin receptor and lysosomal proteins accumulated at the cell surface upon overexpression of chimeric proteins containing functional YXXO targeting signals, regardless of the compartmental destination imparted by the signal. Furthermore, overexpression of these chimeric proteins compromised YXXO-mediated endocytosis and lysosomal delivery. These activities were ablated by mutating the signals or by appending sequences that conformed to the YXXO motif but lacked targeting activity. Interestingly, overexpression of chimeric proteins containing cytoplasmic LL signals failed to induce surface displacement of endogenous YXXO-containing proteins, but did displace other proteins containing LL motifs. Our data demonstrate that: (a) Protein targeting and internalization mediated by either YXXO or LL motifs are saturable processes; (b) common saturable components are used in YXXO-mediated protein internalization and targeting to different post-Golgi compartments; and (c) YXXO- and LL-mediated targeting mechanisms use distinct saturable components.     

Two lysosomal membrane proteins,LGP85 and LGP107, are delivered to late endosomes/lysosomes through different intracellular routes after exiting from the trans-Golgi network

Lysosomal membrane proteins are delivered from their synthesis site, the endoplasmic reticulum (ER) to late endosomes/lysosomes through the Golgi complex. It has been proposed that after leaving the Golgi they are transported either directly or indirectly (via the cell surface) to late endosomes/lysosomes. In the present study, we examined the transport routes taken by two structurally different lysosomal membrane proteins, LGP85 and LGP107, in rat 3Y1-B cells. Here we show that newly synthesized LGP85 and LGP107 are delivered to late endosomes/lysosomes via a direct route without passing through the cell surface. Interestingly, although LGP107 is delivered from the Golgi to early endosomes containing internalized horseradish peroxidase-conjugated transferrin (HRP-Tfn) en route to lysosomes, LGP85 does not pass through the HRP-Tfn-positive early endosomes. These results suggest, therefore, that LGP85 and LGP107 are sorted into distinct transport vesicles at the post-Golgi, presumably the trans-Golgi network (TGN), after which LGP85 is delivered directly to late endosomes/lysosomes, but significant fractions of LGP107 are targeted to early endosomes before transport to late endosomes/lysosomes. This study provides the first evidence that after exiting from the Golgi, LGP85 and LGP107 are targeted to late endosomes/lysosomes via a different pathway.     

P-selectin targeting to secretory lysosomes of Rbl-2H3 cells.

The biogenesis of secretory lysosomes, which combine characteristics of both lysosomes and secretory granules, is currently of high interest. In particular, it is not clear whether delivery of membrane proteins to the secretory lysosome requires lysosomal, secretory granule, or some novel targeting determinants. Heterologous expression of P-selectin has established that this membrane protein contains targeting signals for both secretory granules and lysosomes. P-selectin is therefore an ideal probe with which to determine the signals required for targeting to secretory lysosomes. We have exploited subcellular fractionation and immunofluorescence microscopy to monitor targeting of transiently expressed wild-type and mutant horseradish peroxidase (HRP)-P-selectin chimeras to secretory lysosomes of Rbl-2H3 cells. The exposure of the HRP chimeras to intracellular proteolysis was also determined as a third monitor of secretory lysosome targeting. Our data show that HRP-P-selectin accumulates in secretory lysosomes of Rbl-2H3 cells using those cytoplasmic sequences previously found to be sufficient for targeting to conventional lysosomes. This work highlights the similar sorting signals used for targeting of membrane proteins to conventional lysosomes and secretory lysosomes.     

Mechanisms of chaperone-mediated autophagy

Chaperone-mediated autophagy is one of several lysosomal pathways of proteolysis. This pathway is activated by physiological stresses such as prolonged starvation. Cytosolic proteins with particular peptide sequence motifs are recognized by a complex of molecular chaperones and delivered to lysosomes. No vesicular traffic is required for this protein degradation pathway, so it differs from microautophagy and macroautophagy. Protein substrates bind to a receptor in the lysosomal membrane, the lysosome-associated membrane protein (lamp) type 2a. Levels of lamp2a in the lysosomal membrane are controlled by alterations in the lamp2a half-life as well as by the dynamic distribution of the protein between the lysosomal membrane and the lumen. Substrate proteins are unfolded before transport into the lysosome lumen, and the transport of substrate proteins requires a molecular chaperone within the lysosomal lumen. The exact roles of this lysosomal chaperone remain to be defined. The mechanisms of chaperone-mediated autophagy are similar to mechanisms of protein import into mitochondria, chloroplasts, and the endoplasmic reticulum.     

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.     

Retention of subunits of the oligosaccharyltransferase complex in the endoplasmic reticulum

Membrane proteins of the endoplasmic reticulum (ER) may be localized to this organelle by mechanisms that involve retention, retrieval, or a combination of both. For luminal ER proteins, which contain a KDEL domain, and for type I transmembrane proteins carrying a dilysine motif, specific retrieval mechanisms have been identified. However, most ER membrane proteins do not contain easily identifiable retrieval motifs. ER localization information has been found in cytoplasmic, transmembrane, or luminal domains. In this study, we have identified ER localization domains within the three type I transmembrane proteins, ribophorin I (RI), ribophorin II (RII), and OST48. Together with DAD1, these membrane proteins form an oligomeric complex that has oligosaccharyltransferase (OST) activity. We have previously shown that ER retention information is independently contained within the transmembrane and the cytoplasmic domain of RII, and in the case of RI, a truncated form consisting of the luminal domain was retained in the ER. To determine whether other domains of RI carry additional retention information, we have generated chimeras by exchanging individual domains of the Tac antigen with the corresponding ones of RI. We demonstrate here that only the luminal domain of RI contains ER retention information. We also show that the dilysine motif in OST48 functions as an ER localization motif because OST48 in which the two lysine residues are replaced by serine (OST48ss) is no longer retained in the ER and is found instead also at the plasma membrane. OST48ss is, however, retained in the ER when coexpressed with RI, RII, or chimeras, which by themselves do not exit from the ER, indicating that they may form partial oligomeric complexes by interacting with the luminal domain of OST48. In the case of the Tac chimera containing only the luminal domain of RII, which by itself exits from the ER and is rapidly degraded, it is retained in the ER and becomes stabilized when coexpressed with OST48.     

Sequence requirements for ligand binding and cell surface expression of the Tac antigen, a human interleukin-2 receptor

Discrete peptide domains within the primary sequence of cell-surface receptor glycoproteins are believed to regulate not only their function but also their targeting to the cell membrane. To identify sequence elements required for intracellular transport and ligand binding by the human Tac interleukin-2 (IL-2) receptor, we prepared expression plasmids encoding a series of artificially mutated or naturally occurring variants of the Tac cDNA. In particular, we sought to further delineate the functional role of the sequences contributed by each of the eight exons that together encode the Tac protein. Deletion of exons 5 through 8 of the receptor had no detectable effect on IL-2 binding or intracellular transport of the Tac protein, and resulted in secreted forms of this IL-2-binding protein. Removal of sequences corresponding to all of exon 4 ablated IL-2 binding activity yet still permitted transport to the cell surface. In contrast, partial deletion of exon 4 sequences resulted in proteins that not only lacked IL-2 binding activity but also were sequestered within the endoplasmic reticulum. Removal of one or both of the N-linked glycosylation sites present in the Tac protein did not impair receptor transport or ligand binding. These results demonstrate that exon 4 of the Tac gene encodes amino acid residues that play an important role in regulating both the intracellular transport and function of this IL-2 receptor.     

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.     

Lysosomal localization of TRPML3 depends on TRPML2 and the mucolipidosis-associated protein TRPML1

Mucolipidosis type IV is an autosomal recessive lysosomal storage disorder characterized by severe neurodegeneration, achlorhydria, and visual impairments such as corneal opacity and strabismus. The disease arises due to mutations in a group 2 transient receptor potential (TRP)-related cation channel, TRPML1. Mammals encode two additional TRPML proteins named TRPML2 and TRPML3. Information regarding the propensity of these proteins to multimerize, their subcellular distribution and mechanisms that regulate their trafficking are limited. Here we demonstrate that TRPMLs interact to form homo- and heteromultimers. Moreover, the presence of either TRPML1 or TRPML2 specifically influences the spatial distribution of TRPML3. TRPML1 and TRPML2 homomultimers are lysosomal proteins, whereas TRPML3 homomultimers are in the endoplasmic reticulum. However, TRPML3 localizes to lysosomes when coexpressed with either TRPML1 or TRPML2 and is comparably mislocalized when lysosomal targeting of TRPML1 and TRPML2 is disrupted. Conversely, TRPML3 does not cause retention of TRPML1 or TRPML2 in the endoplasmic reticulum. These data demonstrate that there is a hierarchy controlling the subcellular distributions of the TRPMLs such that TRPML1 and TRPML2 dictate the localization of TRPML3 and not vice versa.     

The intracellular trafficking of opioid receptors directed by carboxyl tail and a di-leucine motif in Neuro2A cells

The mu- and delta-opioid receptors (MOR and DOR) differ significantly in their intracellular trafficking. MORs recycle back to the cell surface upon agonist treatment, whereas most internalized DORs are targeted to lysosomes for degradation. By exchanging the carboxyl tail domains of MOR and DOR and expressing the receptor chimeras in mouse neuroblastoma Neuro2A cells, it could be demonstrated that the carboxyl tail domain is not the sole determinant in directing the intracellular trafficking in these Neuro2A cells. Deletion of the dileucine motif (Leu245-Leu246) within the third intracellular loop of DOR or the mutation of Leu245 to Met slowed the lysosomal targeting of these delta-opioid receptors. Meanwhile the mutation of Met264 to Leu increased the rate of agonist-induced receptor internalization and the lysosomal targeting of the wild type and the delta-opioid receptor carboxyl tail chimera of the mu-opioid receptor. These studies suggest interplay between a di-leucine motif and the carboxyl tail in the lysosomal targeting of the receptor.     

Colocalized transmembrane determinants for ER degradation and subunit assembly explain the intracellular fate of TCR chains

The intracellular fate of T cell antigen receptor (TCR) subunits (alpha beta gamma delta epsilon zeta 2) is determined by their assembly in the endoplasmic reticulum (ER). To study the structural bases for this tight correlation between assembly and intracellular fate, we sought to define the nature of determinants for both ER degradation and subunit assembly within the TCR-alpha chain. We found that a 9 amino acid transmembrane sequence of the TCR-alpha chain, containing 2 critical charged residues, was sufficient to cause ER degradation when placed in the context of the Tac antigen, used here as a reporter protein. CD3-delta assembled with chimeric proteins containing this short transmembrane sequence, and this assembly resulted in abrogation of targeting for ER degradation. Thus, the colocalization of determinants for ER degradation and sites of subunit interactions explains how the fate of some newly synthesized TCR chains can be decided on the basis of their assembly status.     

Selective degradation of annexins by chaperone-mediated autophagy

Annexins are a family of proteins that bind phospholipids in a calcium-dependent manner. Analysis of the sequences of the different members of the annexin family revealed the presence of a pentapeptide biochemically related to KFERQ in some annexins but not in others. Such sequences have been proposed to be a targeting sequence for chaperone-mediated autophagy, a lysosomal pathway of protein degradation that is activated in confluent cells in response to removal of serum growth factors. We demonstrate that annexins II and VI, which contain KFERQ-like sequences, are degraded more rapidly in response to serum withdrawal, while annexins V and XI, without such sequences, are degraded at the same rate in the presence and absence of serum. Using isolated lysosomes, only the annexins containing KFERQ-like sequences are degraded by chaperone mediated-autophagy. Annexins V and XI could associate with lysosomes but did not enter the lysosomes and were not proteolytic substrates. Furthermore, four annexins containing KFERQ-like sequences, annexins I, II, IV, and VI, are enriched in lysosomes with high chaperone-mediated autophagy activity as expected for substrate proteins. These results provide striking evidence for the importance of KFERQ motifs in substrates of chaperone-mediated autophagy.     

Trafficking of lysosomal enzymes

The targeting of lysosomal enzymes from their site of synthesis in the rough endoplasmic reticulum (RER) to their final destination in lysosomes is directed by a series of protein and carbohydrate recognition signals on the enzymes. Lysosomal enzymes, along with secretory and plasma membrane proteins, contain amino-terminal signal sequences that direct the vectorial discharge of the nascent proteins into the lumen of the RER. The three classes of proteins also share a common peptide signal for asparagine glycosylation. The next signal is unique to lysosomal enzymes and permits their high-affinity binding to a specific phosphotransferase that catalyzes the formation of the mannose 6-phosphate recognition marker. This carbohydrate determinant allows binding to specific receptors that translocate the lysosomal enzymes from the Golgi complex to an acidified prelysosomal compartment. There the lysosomal enzymes are discharged for final packaging into lysosomes. Two distinct mannose 6-phosphate receptors have been identified, and cDNAs encoding their entire sequences have been cloned. An analysis of the deduced amino acid sequences of the receptors shows that each is composed of four structural domains: a signal sequence, an extracytoplasmic amino-terminal domain, a hydrophobic membrane-spanning region, and a cytoplasmic domain. The entire extracytoplasmic region of the small receptor is homologous to the 15 repeating domains that constitute the extracytoplasmic portion of the large receptor.     

A complex web of signal-dependent trafficking underlies the triorganellar distribution of P-selectin in neuroendocrine PC12 cells.

By analyzing the trafficking of HRP-P-selectin chimeras in which the lumenal domain of P-selectin was replaced with horseradish peroxidase, we determined the sequences needed for targeting to synaptic-like microvesicles (SLMV), dense core granules (DCG), and lysosomes in neuroendocrine PC12 cells. Within the cytoplasmic domain of P-selectin, Tyr777 is needed for the appearance of P-selectin in immature and mature DCG, as well as for targeting to SLMV. The latter destination also requires additional sequences (Leu768 and 786DPSP789) which are responsible for movement through endosomes en route to the SLMV. Leu768 also mediates transfer from early transferrin (Trn)-positive endosomes to the lysosomes; i.e., operates as a lysosomal targeting signal. Furthermore, SLMV targeting of HRP-P-selectin chimeras, but not the endogenous SLMV protein synaptophysin/p38, previously shown to be delivered to SLMV directly from the plasma membrane, is a Brefeldin A-sensitive process. Together, these data are consistent with a model of SLMV biogenesis which involves an endosomal intermediate in PC12 cells. In addition, we have discovered that impairment of SLMV or DCG targeting results in a concomitant increase in lysosomal delivery, illustrating the entwined relationships between routes leading to regulated secretory organelles (RSO) and to lysosomes.     

Different Steady State Subcellular Distributions of the Three Splice Variants of Lysosome-associated Membrane Protein LAMP-2 Are Determined Largely by the COOH-terminal Amino Acid Residue

The extensively glycosylated lysosome-associated membrane proteins (LAMP)-2a, b, and c are derived from a single gene by alternative splicing that produces proteins with differences in the transmembrane and cytosolic domains. The lysosomal targeting signals reside in the cytosolic domain of these proteins. LAMPs are not restricted to lysosomes but can also be found in endosomes and at the cell surface. We investigated the subcellular distribution of chimeras comprised of the lumenal domain of avian LAMP-1 and the alternatively spliced domains of avian LAMP-2. Chimeras with the LAMP-2c cytosolic domain showed predominantly lysosomal distribution, while higher levels of chimeras with the LAMP-2a or b cytosolic domain were present at the cell surface. The increase in cell surface expression was due to differences in the recognition of the targeting signals and not saturation of intracellular trafficking machinery. Site-directed mutagenesis defined the COOH-terminal residue of the cytosolic tail as critical in governing the distributions of LAMP-2a, b, and c between intracellular compartments and the cell surface.     

Core-glycosylated mucin-like repeats from MUC1 are an apical targeting signal

MUC1 is efficiently delivered to the apical surface of polarized Madin-Darby canine kidney (MDCK) cells by transit through apical recycling endosomes, a route associated with delivery of apical proteins with glycan-dependent targeting signals. However, a role for glycans in MUC1 sorting has not been established. A key feature of MUC1 is a heavily O-glycosylated mucin-like domain with a variable number of nearly perfect tandem repeats and adjacent imperfect repeats. Metabolic labeling, cell surface biotinylation, immobilized lectins, and confocal immunofluorescence microscopy were used to characterize the polarized delivery of MUC1 mutants and chimeras in MDCK cells to identify the apical targeting signal. Both the interleukin-2 receptor α subunit (Tac) and a chimera where the Tac ectodomain replaced that of MUC1 were delivered primarily to the basolateral surface. Attachment of the MUC1 mucin-like domain to the N terminus of Tac enhanced apical but not basolateral delivery when compared with Tac. Conversely, deletions within the mucin-like domain in MUC1 reduced apical but not basolateral delivery when compared with MUC1. In pull-down assays with lectins, we found a notable difference in the presence of core 1 O-glycans, but not poly-N-acetyllactosamine, in apically targeted MUC1 and chimeras when compared with Tac. Consistent with these data, we found no effect on MUC1 targeting when galectin-3, with preference for poly-N-acetyllactosamine, was depleted from polarized MDCK cells. However, we did block the apical targeting activity of the mucin-like repeats when we overexpressed CMP-Neu5Ac:GalNAc-Rα2,6-sialyltransferase-1 to block core O-glycan synthesis. The cumulative data indicate that the core-glycosylated mucin-like repeats of MUC1 constitute an apical targeting signal.     

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.