The C-terminal cytoplasmic tail of mucolipin-1 is sufficient for targeting to early endosomes
To assess whether targeting information is present in the C-terminal cytoplasmic tail of mucolipin-1, a chimeric protein was created in which the last 63 residues of mucolipin-1 were appended to the lumenal and transmembrane domains of Tac, a cell-surface type 1 transmembrane glycoprotein that has been used previously in chimeric constructs to analyze carboxyl-terminal cytoplasmic targeting signals (22
). The localization of the chimeric protein, termed Tac-MLN-CTail (for Tac lumenal-Tac transmembrane-Mucolipin-1 C-terminal cytoplasmic tail), was compared with that of intact Tac by indirect immunofluorescence microscopy in HeLa cells using antibodies directed to the Tac lumenal domain. shows that although Tac was distributed mostly at the cell surface with very little in internal structures, Tac-MLN-CTail was present mostly in vesicles scattered through the cytoplasm.
The C-terminal tail of mucolipin-1 contains sorting information for location to early endosomes.
To further characterize the compartment to which the C-terminal tail of mucolipin-1 targeted the Tac antigen, we performed double staining by indirect immunofluorescence analysis to co-localize Tac-MLN-CTail with various endogenous and transfected molecules. As seen in , the Tac-MLN-CTail chimera showed a clear co-localization with early endosomal markers, such as internalized transferrin, Hrs, Rab5-green fluorescent protein (Rab5-GFP) or its constitutively active version, Rab5-Q79L-GFP. Conversely, no co-localization was observed with late endosomal or lysosomal markers (Supplementary Material, Figure S1). It is interesting to note that the over-expression of Tac-MLN-CTail resulted in swollen endosomes. However, this enlargement did not seem to affect the trafficking of EGF or transferrin (our unpublished results).
Identification of a targeting motif in the C-terminal cytosolic tail of mucolipin-1
It has been previously described that most lysosomal proteins have short cytosolic tails containing tyrosine or di-leucine-based motifs that determine their lysosomal targeting (23
). The analysis of the C-terminal cytoplasmic tail of mucolipin-1 revealed the presence of several putative sorting motifs. These include a YXX
DTI), a di-leucine motif (L563
L) and an acidic di-leucine motif (D/E)XXXL(L/I) (E573
EHSLL) (). To determine whether these or other signals were responsible for targeting Tac-MLN-CTail to endosomes, we mutated the three motifs individually by substitution of key residues to alanines, and the resulting immunofluorescence profiles were analyzed in transiently transfected HeLa cells. shows that the mutation of Leu577 and Leu578 to alanines (Tac-MLN-CTail-L577
L/AA) caused the protein to be retained at the plasma membrane, whereas the substitution of Tyr521 (Tac-MLN-CTail-Y521
/A) or Leu563 (Tac-MLN-CTail-L563
/A) with alanines had no effect on the distribution of the chimeras.
A di-leucine motif located at C-terminal tail of mucolipin-1 mediates internalization of Tac-MLN-CTail chimera.
To determine whether the (D/E)XXXL(L/I) motif acts as a signal for internalization from the cell surface, we performed antibody-uptake experiments. Antibodies to Tac were bound on ice to the surface of HeLa cells expressing Tac, Tac-MLN-CTail or Tac-MLN-CTail-L577L/AA, then bound antibody was allowed to internalize for 15 min at 37 °C, and the amount of protein remaining at the cell surface was quantified by flow cytometry (FACS). As seen in , nearly 60% of the antibody initially bound to Tac-MLN-CTail was internalized by 15 min. In contrast, no internalization was observed for the Tac-MLN-CTail-L577L/AA chimera. These data indicate that the E573EHSLL sequence is an essential component of the endosomal targeting signal in mucolipin-1 cytoplasmic tail.
Endocytosis of Tac-MLN-CTail is dependent on clathrin adaptor AP2
It has been extensively studied that (D/E)XXXL(L/I) motifs are recognized with high specificity by the AP complexes. Four different AP complexes have been described and termed AP1, AP2, AP3 and AP4. Although all four complexes participate in cargo selection and vesicle formation in the endocytic and secretory pathways, they exhibit distinct intracellular localizations. AP2 is exclusively located at the plasma membrane and plays an essential role in the endocytosis of specific cargo proteins. To address whether AP2 participates in the internalization of the Tac-MLN-CTail chimera, we reduced the expression of endogenous AP2 in HeLa cells using specific siRNA oligonucleotides. Immunoblot analysis revealed a 90% reduction in the levels of AP2 when compared with cells transfected with control (non-silencing) siRNA (). The levels of annexin-II were monitored as control for the specificity of the antisense. As expected, HeLa cells depleted of AP2 showed very little internalization of rhodamine-transferrin when compared with control cells, indicating that AP2-dependent endocytosis is impaired (). Likewise, FACS analysis revealed that the internalization of Tac-MLN-CTail was blocked in the absence of AP2. shows that in cells treated with control non-silencing siRNA approximately 35% of Tac-MLN-CTail remained at the plasma membrane after 15 min internalization. In contrast, almost no endocytosis of Tac-MLN-CTail was observed in cells depleted of AP2. In addition, surface staining of Tac-MLN-CTail showed a good co-localization between the chimera and AP2-GFP in structures that resemble clathrin-coated-pits (). Altogether, these results indicate that the E573EHSLL sequence can mediate the internalization of Tac-MLN-CTail through an AP2-dependent pathway.
Endocytosis of Tac-MLN-CTail is dependent on clathrin adaptor AP2.
Palmitoylation mediates the association of the C-terminal tail of mucolipin-1 with membranes
We found that the C-terminal cytosolic tail of mucolipin-1 is sufficient to promote the recruitment of GFP to intracellular membranes. As seen in Supplementary Material, Figure and , the expression of a chimera containing the C-terminal tail of mucolipin-1 fused to GFP (GFP-MLN-CTail) showed staining of the plasma membrane and cytosolic vesicles. The vesicles strongly co-localized with early endosomal markers such as internalized transferrin, EEA1 and Hrs, as well as with Tac-MLN-CTail (Supplementary Material, Figure S2). These data indicate that the C-terminal tail of mucolipin-1 has the ability to associate with membranes independently of the presence of a transmembrane domain, suggesting that acylation may be responsible for the observed subcellular targeting.
Palmitoylation of three cysteine residues mediates recruitment of GFP-MLN-CTail to membranes.
Our examination of the sequence of the mucolipin-1 C-terminal tail revealed the presence of four cysteines (C561
) that appear to be likely candidates to undergo palmitoylation via thioester linkages. To assess whether palmitoylation might be responsible for the association of the mucolipin-1 C-terminal tail with intracellular membranes, we incubated HeLa cells expressing GFP-MLN-CTail for 12 h with 2-bromopalmitate (2-BP), a potent inhibitor of protein palmitoylation (24
). As predicted, in cells treated with 2-BP, GFP-MLN-CTail exhibited no detectable membrane staining but instead displayed a diffuse cytoplasmic staining (). Next, we carried out systematic mutagenesis of the C561
SLLCCC sequence by individually substituting each of these residues with alanines and analyzing the location of the resulting chimeras by confocal microscopy. The results are summarized in Interestingly, the substitution of the three cysteine residues located between positions 565 and 567 with alanines (GFP-MLN-CTail-C565
CC/AAA) abolished the ability of the C-terminal tail of mucolipin-1 to associate with membranes (; ). Individual mutation of each of these three cysteines revealed that Cys566 is essential for recruitment to membranes, whereas the mutation of Cys565 or Cys567 had only a partial effect on the ability of the chimeras to interact with intracellular membranes. The mutation of Leu564 to alanine also caused redistribution to the cytosol, suggesting that this amino acid is required for optimal palmitoylation. In contrast, the substitution of Cys561, Ser562 or Leu563 with alanine did not affect the distribution of the chimeras. Interestingly, a mutant that lacks the last 13 residues but still contains Cys565, Cys566 and Cys567 retained its ability to associate with membranes but was localized exclusively to the plasma membrane. Similarly, the substitution of Leu577 and Leu578 with alanine also resulted in the redistribution of chimera to the plasma membrane (). These data suggest that the C565
CC sequence is required for palmitoylation and subsequent association with membranes although the acidic di-leucine motif mediates localization at endosomes.
Multinational analysis of the C-terminal tail of mucalipin-1
To further confirm the role of palmitoylation on the membrane partitioning of the GFP chimeras, we fractionated cellular lysates from cells expressing the above chimeras into cytosolic (C) and membrane (M) fractions through high-speed ultracentrifugation. shows that approximately 50% of GFP-MLN-CTail partitioned into the membrane-associated fractions, whereas GFP-MLN-CTail-C565CC/AAA was predominantly soluble. The treatment of cells expressing GFP-MLN-CTail with 2-BP also caused the redistribution of chimera to the soluble fraction. In comparison, non-palmitoylated Rab5 localization was not altered by 2-BP treatment, indicating that the drug is specific in blocking modification by palmitate (data not shown). These results corroborate that preventing palmitoylation by cysteine-to-alanine mutations or 2-BP treatment blocks the ability of the C-terminal tail of mucolipin-1 to stably bind to membranes.
Palmitoylation regulates the efficiency of internalization of Tac-MLN-CTail
It has been described that the position of sorting motifs relative to the transmembrane domain may influence the affinity of the interaction with the sorting machinery (23
). In the case of endocytic signals, they are often (although not always) situated at proximity from the membrane. This proximity could facilitate the interaction of AP2 with specific lipids such as phosphatidylinositol 4, 5-biphosphate (PIP2) or other PIP2-binding proteins. Interestingly, the proximity of a sorting motif to the membrane may be shortened by palmitoylation. It has been reported that the palmitoylation of specific cysteine residues in the cytosolic tail of the cation-dependent mannose 6-phosphate receptor may serve to anchor this domain closer to the membrane by insertion of the palmitoyl chains into the lipid bilayer (25
Consistent with this idea, we hypothesized that palmitoylation of the C565CC site could help bring the E573EHSLL signal closer to the plasma membrane, thus facilitating endocytosis. To address this possibility, we compared the efficiency of internalization of Tac, Tac-MLN-CTail and Tac-MLN-CTail-C565CC/AAA by FACS. As shown in , the percent of Tac-MLN-CTail-C565CC/AAA that is internalized after 15 min was reduced to almost half when compared with that of Tac-MLN-Ctail (35% versus 61%). Together, our results suggest that the palmitoylation of the C-terminal tail of mucolipin-1 may play a role in regulating its trafficking possibly through bringing the endocytic signal closer to the plasma membrane, thus increasing the efficiency of endocytosis.
Effect of palmitoylation on the internalization of Tac-MLN-CTail.
To test whether full-length mucolipin-1 was palmitoylated, we transfected mucolipin-1 wild-type (MLN-GFP; see below) as well as a mutant lacking the putative palmitoylated cysteines (MLN-GFP-C565CC/AAA) into HeLa cells and metabolically labeled these cells with [3H]palmitate followed by immunoprecipitation and SDS-PAGE. As seen in , the immunoprecipitation of full-length mucolipin-1 revealed a band of approximately 90 kDa that corresponds to the predicted molecular weight of the chimera and was labeled with [3H]palmitate. In contrast, no metabolic labeling was observed for the MLN-GFP-C565CC/AAA mutant. Therefore, we conclude that mucolipin-1 is palmitoylated in vivo and that Cys565, Cys566 and Cys567 account for the total palmitoylation of the protein.
Full-length mucolipin-1 contains additional sorting information that regulates its transport to lysosomes
We next analyzed the distribution of full-length mucolipin-1. To do so, we generated a chimera in which GFP was fused to the C-terminal end of mucolipin-1 (MLN-GFP). When transiently transfected into HeLa cells, MLN-GFP localized in vacuolar structures containing late endosomal–lysosomal markers such as CD63 and lamp-1 (). In contrast, no significant co-localization with early endosomal markers was observed (data not shown). This distribution is consistent with recent data showing that cup-5, the C. elegans
ortholog of mucolipin-1, is present in both mature lysosomes and specific subdomains of late endosomes where endosomal cargo destined for the lysosome accumulates (21
). In addition, Manzoni et al. (26
) have recently described that a myc-epitope tagged version of mucolipin-1 mostly co-localizes with lamp-1. It has been suggested that mucolipin-1 could play an important role in the regulation of lysosomal biogenesis. In agreement with this idea, we found that the over-expression of MLN-GFP in HeLa cells caused a dramatic enlargement of lysosomes. These structures resemble the late endosome–lysosome hybrid organelles observed in cup-5-defective cells (see arrows in ). Interestingly, very little co-localization was observed between Tac-MLN-CTail and MLN-GFP, indicating that additional sorting information, located in a region different from the C-terminal tail, mediates the transport of the full-length mucolipin-1 to lysosomes.
Full-length mucolipin-1 contains additional sorting signals that regulate its transport to lysosomes.
The N-terminal cytoplasmic tail of mucolipin-1 contains targeting information for sorting to lysosomes
The search for sorting sequences that mediate the delivery of mucolipin-1 to lysosomes revealed the presence of a (D/E)XXXL(L/I) motif (E11TERLL) at the N-terminal cytosolic tail of the protein that could potentially mediate interaction with APs and transport to lysosomes. To determine whether this motif is in fact functional, we fused the N-terminal tail of mucolipin-1 (residues from positions 1 to 66) to the extracellular and transmembrane domains of Tac, and the distribution of the resulting chimera (Tac-MLN-NTail) was analyzed by confocal microscopy. As shown in , Tac-MLN-NTail localized predominantly to large vesicular structures within the cytoplasm. To determine the nature of these vesicles, we monitored EGF uptake using EGF-Alexa Fluor 555. EGF binds to its receptor on the cell surface, and receptor–ligand complexes are internalized by clathrin-coated vesicles and transported to lysosomes for degradation. After uptake for 30 min, EGF labeled small vesicles throughout the cytoplasm that correspond to early and/or late endosomes and did not contain Tac-MLN-NTail. In contrast, after 3 h of internalization, EGF accumulated at lysosomes and showed good co-localization with Tac-MLN-NTail. Extensive co-localization was also observed between Tac-MLN-NTail and MLN-GFP. These results indicate that the N-terminal tail of mucolipin-1 contains sorting information for transport to lysosomes.
The N-terminal tail of mucolipin-1 contains sorting information for transport to lysosomes.
To be sure that the cloning of the N-terminal tail fused to Tac in a ‘type I’ orientation (instead of the ‘type II’ conformation that this fragment has in mucolipin-1) was not affecting the sorting of the chimera, we generated a construct in which the NTail of mucolipin-1 was fused to the transmembrane and extracellular domains of the transferrin receptor (TfR). Our data showed that the MLN-NTail-TfR-GFP chimera is partially delivered to lysosomes (as seen by co-localization with lamp-1), whereas wild-type TfR-GFP localizes at early endosomes and plasma membrane (data not shown). These results corroborate the presence of lysosomal sorting motifs at the N-terminal tail and indicate that the transport of Tac-MLN-NTail to lysosomes is not a consequence of the ‘reverse’ orientation of the chimera.
One interesting observation from our previous experiment was that Tac-MLN-NTail could only be detected after incubation of the cells with the lysosomal inhibitor leupeptin. This suggests that after the synthesis of the protein, Tac-MLN-NTail is rapidly transported to lysosomes for degradation. To test this hypothesis, we followed the fate of newly synthesized Tac-MLN-NTail by performing ‘pulse–chase’ experiments by treatment with Brefeldin A (BFA). This approach has been recently used to monitor the transport of LEP100 and CD63 to lysosomes (27
). HeLa cells were transfected with the Tac-MLN-NTail or the Tac-MLN-CTail chimera, and 6 h after transfection, the cells were incubated with 2 µg/mL of BFA for 12 h to accumulate the newly synthesized proteins in the endoplasmic reticulum (ER) (28
). Proteins were then released from the ER by incubation in medium without BFA, and cells were analyzed at different time-points. We found that, whereas the levels of Tac-MLN-CTail remained stable, most of Tac-MLN-NTail labeling disappeared after a 4-h chase. As expected, the degradation of Tac-MLN-NTail was inhibited in the presence of leupeptin, indicating that the degradation of the protein occurs at lysosomes ().
It is important to note that during the BFA ‘pulse–chase’ experiments we observed that most of Tac-MLN-CTail reaches the plasma membrane before its location at early endosomes. In contrast, we did not detect Tac-MLN-NTail at the plasma membrane at any time, suggesting that the chimera travels directly from the TGN to the endosomal–lysosomal system. In agreement with this idea, the distribution of Tac-MLN-NTail did not change in cells treated with siRNA against AP2, thus suggesting that the chimera does not travel via the plasma membrane en route to lysosomes. In contrast, most of Tac-MLN-CTail accumulated at the plasma membrane in the absence of AP2 (Supplementary Material, Figure).
To determine whether Tac-MLN-NTail passes through early endosomes, we co-expressed the Tac-MLN-NTail chimera together with the constitutively active form of Rab5 (Rab5-Q79L-GFP). It is known that the over-expression of Rab5-Q79L results in enlarged endosomes and interferes with both receptor-mediated and fluid-phase endocytosis (29
). As seen in , the over-expression of Rab5-Q79L caused the retention of Tac-MLN NTail in early endosomes allowing the detection of the chimera for long periods of time in the absence of leupeptin.
Mutation of a di-leucine motif located at N-terminal tail of mucolipin-1 causes mistargeting of a Tac-MLN-NTail chimera to the plasma membrane.
Finally, we addressed whether the (D/E)XXXL(L/I) motif located at the N-terminal tail is responsible for the targeting of Tac-MLN-NTail to lysosomes. As seen in , the mutation of Leu15 and Leu16 to alanines (Tac-MLN-NTail-L15L/AA) abolished the delivery of the resulting chimera to lysosomes, instead causing it to accumulate predominantly at the plasma membrane. These results indicate that the E11TERLL sequence acts as a lysosomal targeting motif regulating the transport of the Tac-MLN-NTail protein from the TGN to lysosomes via early endosomes.
Two di-leucine motifs regulate the transport of mucolipin-1 to lysosomes
The data described above allowed us to conclude the existence of two different sorting motifs, an internalization motif and a lysosomal targeting motif located at the C- and N-terminal tails, respectively. To determine whether these sequences are functional in the context of the full-length protein or whether one of the motifs is preferred, we mutated each of the di-leucine pairs and analyzed the distribution of the resulting proteins by confocal microscopy. shows that the mutation of neither Leu15Leu16 (MLN-GFP-L15L/AA) nor Leu577Leu578 (MLN-GFP-L577L/AA) pair to alanines prevented MNL-GFP from reaching lysosomes (as seen by co-localization with lamp-1, Supplementary Material, Figure S4). However, simultaneous mutation of both di-leucine pairs (MLN-GFP-L15L/AA-L577L/AA) resulted in the accumulation of MLN-GFP at the plasma membrane.
Both di-leucine motifs are required for targeting of mucolipin-1 to lysosomes.
Interestingly, the over-expression of Rab5-Q79L-GFP caused a very clear accumulation of MLN-RFP, as well as MLN-RFP-L15L/AA and MLN-RFP-L577L/AA, into Rab5-Q79L-GFP-positive structures indicating that, independently of the route used, mucolipin-1 travels through early endosomes on its way to lysosomes ().
Finally, we addressed whether palmitoylation plays a role in the sorting of full-length mucolipin-1. Data showed that the mutation of the palmitoylation signal alone (MLN-GFP-C565CC/AAA) did not have a clear effect on the trafficking of the protein, as the distribution of MLN-GFP-C565CC/AAA resembled that of MLN-GFP. However, the mutation of Cys565, Cys566 and Cys567 in the MLN-GFP-L15L/AA mutant (MLN-GFP-L15L/AA-C565CC/AAA) or the treatment of MLN-GFP-L15L/AA with 2-BP caused a clear accumulation of the chimeras at the plasma membrane (Supplementary Material, Figure).
In conclusion, our results indicate that either one of the di-leucine motifs can mediate the sorting of mucolipin-1 to lysosomes and reveal a role for palmitoylation in the regulation of the protein trafficking.