Role of protein kinase D in Golgi exit and lysosomal targeting of the transmembrane protein, Mcoln1

Abstract

The targeting of lysosomal transmembrane proteins from the Golgi apparatus to lysosomes is a complex process that is only beginning to be understood. Here, the lysosomal targeting of Mcoln1, the transmembrane protein defective in the autosomal recessive disease, Mucolipidosis, type IV, was studied by over-expressing full length and truncated forms of the protein in human cells, followed by detection using immunofluorescence and immunoblotting. We demonstrated that a 53 amino acid C-terminal region of Mcoln1 is required for efficient exit from the Golgi. Truncations lacking this region exhibited reduced delivery to lysosomes and decreased proteolytic cleavage of Mcoln1 into characteristic ~35 kDa fragments, suggesting that this cleavage occurs in lysosomes. In addition, we found that co-expression of full length Mcoln1 with kinase-inactive protein kinase D (PKD) 1 or 2 inhibited Mcoln1 Golgi exit and transport to lysosomes and decreased Mcoln1 cleavage. These studies suggest that PKDs play a role in the delivery of some lysosomal resident transmembrane proteins from the Golgi to the lysosomes.

INTRODUCTION

The biogenesis of lysosomes entails the delivery of membrane proteins, lipid membranes and soluble proteins from the Golgi appparatus to endosomal compartments. While the transport of many soluble hydrolases from the Golgi to lysosomes via mannose-6-phosphate receptors is well understood, the mechanisms underlying the delivery of transmembrane (TM) proteins to the lysosomes are complex and only beginning to be elucidated (1). For example, TM proteins may be first delivered to the PM and then travel to lysosomes via the endosomal system, or be transported directly from the Golgi to early endosomes or late endosomes en route to the lysosomes (2). A key element in directing TM proteins to the lysosomes is the presence of cytosolically-oriented amino acid motifs that are recognized by adaptor proteins which target cargo to clathrin-coated transport intermediates at the trans-Golgi network (TGN). For example, DXXLL acidic cluster dileucine motifs bind to GGAs (Golgi localized, γ-ear ARF-binding proteins), whereas [DE]XXXL[LI] and YXXØ (where Ø is any bulky hydrophobic amino acid) sequences are recognized by the heterotetrameric adaptor protein-1 (AP1) complex (3). GGAs and AP1 have been shown to interact; thus it is currently not known if GGAs and AP1 act in concert or participate in two parallel sorting pathways at the TGN (4). Other adaptor protein complexes are also involved in the recognition of the [DE]XXXL[LI] and YXXØ motifs. For example, adaptor protein complex 2 (AP2) mediates clathrin-dependent internalization from the PM of proteins (e.g., lysosomal associated membrane proteins [LAMPs]) containing such motifs (5), whereas adaptor protein complex 3 (AP3) is believed to mediate the transport of some YXXØ motif-containing proteins (e.g., CD63, endolyn) from EE to LE (6).

Another step in lysosomal targeting is the fission of carrier vesicles from the TGN. Some carrier vesicles are reported to arise from membrane fission at the TGN via the action by protein kinase D (PKD) isoforms 1–3 (7). PKDs are thought to be involved in the release of transport vesicles destined for the plasma membrane, specifically proteins targeted to the basolateral membrane in polarized cells (8). PKDs are reported not to be involved in the delivery of the endosomal/lysosomal protein, H2-M, to the late endosomal system (9); however, a recent study demonstrated that PKD3 may play a role in delivering vesicle-associated membrane protein-2 (VAMP2) to its endosomal compartment (10). Thus, a general role for PKDs in the transport of lysosomal proteins is still uncertain.

Mucolipin-1 (Mcoln1) is a six transmembrane, lysosomal protein (11, 12) of the transient receptor potential superfamily that is mutated in the autosomal, recessive neurodegenerative disease, mucolipidosis, type IV (13–15). Mcoln1 is reported to act as channel permeable to Ca²⁺, Fe²⁺ and other ions (16, 17) and posesses a serine lipase consensus domain (13, 18). Further, the absence of Mcoln1 results in lysosomal storage of lipids, mucopolysaccharides and glycoproteins, and altered lysosomal transport (12, 19–22); however, the precise function of Mcoln1 is not known.

A number of investigations have been performed concerning the mechanisms involved in Mcoln1 targeting to the lysosome; however, the results have been somewhat contradictory. Mcoln1 possesses a [DE]XXXL[LI] motif near its N-terminus (ETERLL) and its C-terminus (EEHSLL). While Venkatachalam et al. reported that loss of the C-terminal dileucine motif of Mcoln1 resulted in loss of lysosomal targeting and accumulation at the PM (23), others have demonstrated that mutation of both the N and C-terminal dileucine motifs are needed to disrupt the lysosomal targeting of Mcoln1 (12, 24). Additionally, Vergrajauregui et al. demonstrated that a chimeric protein containing the C-terminal region of Mcoln1 was endocytosed via AP2 dependent mechanisms and accumulated in early endosomes, whereas a chimera containing the N-terminus of Mcoln1 accumulated in lysosomes and was transported by AP-2 independent mechanisms (24). In contrast, Miedel et al. showed that the lysosomal targeting of full length, epitope-tagged Mcoln1 was independent of AP2, but that AP1 knockdown caused accumulation of Mcoln1 in the Golgi apparatus and lack of delivery to lysosomes (25). Thus, questions remain concerning the involvement of adaptor protein complexes in Mcoln1 trafficking.

It has been reported that after maturation, both endogenous and recombinant Mcoln1 are cleaved from an intact 60–70 kDa protein to two similar sized ~35 kDa fragments (26). The biological function of Mcoln1 cleavage is unknown but Kiselyov et al. suggested that Mcoln1 cleavage may be a mechanism for regulating the duration of its channel function (26). Although Mcoln1 cleavage was initially proposed to occur in lysosomes (26), Miedel et al. suggested that this proteolysis occurred in a prelysosomal compartment, possibly the TGN, based on the persistence of Mcoln1 cleavage in cells depleted of AP1 (25). Thus, the location at which Mcoln1 proteolytic cleavage occurs is controversial. Understanding of where this process occurs may provide insight into Mcoln1 function.

In the current work, we further investigated Mcoln1 Golgi exit and lysosomal targeting by studying the transport of the untagged, full length protein expressed in human cells. We expressed a series of C-terminal truncations of Mcoln1 and identified a C-terminal region preceding the terminal dileucine motif that is important for efficient Golgi exit and lysosomal targeting of Mcoln1. We also demonstrated that when lysosomal targeting is inhibited, the cleavage of Mcoln1 into ~35 kDa fragments is inhibited, suggesting that this Mcoln1 cleavage occurs at the lysosomes. Finally, we showed that expression of kinase-inactive (KI) PKD1 and PKD2 inhibit the lysosomal delivery and cleavage of Mcoln1. These results suggest a role for PKDs in the transport of some lysosomal transmembrane proteins.

RESULTS

Characterization of antibodies against Mcoln1

To study the lysosomal transport of Mcoln1, the sequence for full length human Mcoln1 DNA was inserted into a mammalian transfection vector and expressed in HeLa cells or HSFs. Lysates of transfected HeLa cells were analyzed by immunoblotting with rabbit antibodies directed against N (ML1-N)- or C (ML1-C)-terminal peptide regions of Mcoln1 (Fig. 1A). Each antibody recognized a doublet at ~65 kDa which corresponds to size of full length of Mcoln1 (Fig. 1B). Initial attempt to detect endogenous Mcoln1 in HeLa cell lysates by immunoblotting were not successful. Thus, HeLa cells were cultured for 2 days with leupeptin because a previous report (25) demonstrated that Mcoln1 degradation was inhibited by this treatment. Under such conditions, a triplet was detected in untransfected cells using the ML1-N antibody which ran similarly to the full length forms of over-expressed Mcoln1 (Fig. 1C). From such blots it was calculated that in transfected cells Mcoln1 was over-expressed >80-fold compared to the level of the endogenous protein.

Figure 1. Characterization of antibodies against Mcoln1.

A) Diagram of Mcoln1 showing transmembrane domains, dileucine motifs and antibody epitope regions. Two polyclonal antibodies (ML1-N and ML1-C) were prepared by immunizing rabbits against peptides corresponding to indicated N-and C-terminal regions of Mcoln1. The sites of dileucine motifs and point mutations that cause ER retention of Mcoln1 are also indicated. B) Immunoblotting of wild type Mcoln1 expressed in HeLa cells. Cell lysates were prepared and equal protein amounts (20 μg) were analyzed by SDS-PAGE and immunoblotting. Both ML1-N and ML1-C recognize full length ~65 kDa forms of Mcoln1 as well as ~35 kDa fragments. C) HeLa cells (untransfected or transfected with WT Mcoln1) were cultured in media containing 10 μM leupeptin for 2 days. Cell lysates were prepared and loaded on SDS-PAGE gels using the protein quantities indicated, followed by immunoblotting as above. D) Wild type and mutant Mcoln1 expressed in HeLa cells were analyzed by immunoblotting. L15-16A/L577-578A and L15-16A/G558* are mutants lacking in the N- and C-terminal dileucine motifs and thus are targeted mainly to the PM (see Suppl. Fig. 1). D362Y and L106P are point mutants that are retained in the ER (see E) and show no fragmentation by immunoblotting. Only the lower band of the 65 Kda doublet is present in the ER-retained mutants suggesting that this band represents the immature ER form of Mcoln1. E) Immunofluorescence of wild type and ER-retained mutant Mcoln1 in HeLa cells. HeLa cells were transiently transfected with wild type, D362Y or L106P Mcoln1 as indicated. After 48 h, cells were fixed and processed for immunofluorescence using antibodies against Mcoln1, and either Lamp1 or calnexin. Wild type Mcoln1 closely colocalized with the lysosomal marker, Lamp1. D362Y and L106P mutants colocalized with the ER marker, Calnexin. Bar, 10 μm.

Immunofluorescence detection of wild type Mcoln1 expressed in HeLa cells with these Mcoln1 antibodies demonstrated that this protein was distributed mainly in lysosomes, as evidenced by colocalization with Lamp1 (Fig. 1E). D362Y and L106P mutations of Mcoln1 have been identified in human Mucolipidosis IV patients (15, 27). These mutant Mcoln1 proteins are retained in the endoplasmic reticulum [(26) and Fig. 1E]. Immunoblots of lysates of containing these mutant proteins exhibit only the lower band of the ~65 kDa doublet (Fig. 1D), suggesting that this band represents the ER form of Mcoln1, and allowing us to conclude that the upper band is the mature form of Mcoln1. Both ML1-N and ML1-C antibodies also recognize polypeptides in the 35 kDa range. Previous immunoblotting studies of epitope-tagged forms of Mcoln1 detected similar ~35 kDa fragments of the protein, leading to the suggestion that Mcoln1 is characteristically cleaved into two roughly equally sized products (25, 26).

Identification of a C-terminal region of Mcoln1 required for Golgi exit

Removal of both the N-terminal and C-terminal dileucine motifs in Mcoln1 (mutants L15-16A/L577-578A and L15-16A/G558*) inhibits Mcoln1 lysosomal targeting, and leads to retention of most of the mutant protein at the plasma membrane (Suppl. Fig. 1), as previously reported (12, 24). However, a small portion of these mutant proteins could be detected associated with lysosomes (Suppl. Fig. 1). Immunoblotting of L15-16A/L577-578A and L15-16A/G558* mutants of Mcoln1 show decreased fragmentation compared to wild type Mcoln1 (Fig. 1C). These results suggest that the extent of Mcoln1 cleavage may be linked to the lysosomal delivery of the protein.

To determine if additional regions of Mcoln1 are involved in Golgi exit or lysosomal targeting, C-terminal truncations of Mcoln1 were prepared by site-directed mutagenesis and expressed in HSFs (Fig. 2A). Deletion of the last 22 amino acids from Mcoln1 (mutant G558*) did not lead to any significant alterations in the targeting of Mcoln1 to lysosomes as assessed by colocalization with the lysosomal marker, Lamp1 (Fig. 2B). Truncation at amino acid 540 (Y540*) led to decreased association of the Mcoln1 mutant with Lamp1 (Fig. 2B) and increased association with early endosomes as determined by quantitative colocalization with Lamp1 and EEA1 (Fig. 3A,B). Further truncation of Mcoln just beyond its last transmembrane domain (P527* and Y521*) led to decreased colocalization with Lamp1 (Fig. 2B and Fig. 3B) and an increased retention in perinuclear structures that overlapped with the Golgi marker, GM130 (Fig. 3C). Unlike WT Mcoln1, which colocalized little (~4%) with GM130, mutants P527* and Y521* colocalized 30 and 42%, respectively, with this Golgi marker (Fig. 3D).

Figure 2. Reduced lysosomal localization of Mcoln1 C-terminal truncation mutants.

A) Schematic showing positions of early termination of C-terminal truncation mutants. B) Colocalization of Mcoln1 C-terminal truncation mutants with endogenous Lamp1. Human skin fibroblasts were transiently transfected with WT or mutant Mcoln1. After 48 h, cells were fixed and processed for double label immunofluorescence using antibodies against Mcoln1 and the lysosomal protein, Lamp1. Mcoln1 (red) and Lamp1 (green) signals are merged in the overlay panels at right. Note the progressively reduced colocalization with Lamp1 of increasingly truncated Mcoln1 mutants. Bar, 10 μm.

Figure 3. Altered localization of C-terminal truncation mutants of Mcoln1.

Human skin fibroblasts were transfected with WT or mutant Mcoln1 and processed for immunofluorescence as in Fig. 2. A) Colocalization of Mcoln1 C-terminal truncation mutants with endogenous EEA1, a marker for early endosomes. Note increased colocalization of Y540* Mcoln1 with EEA1 as indicated by arrows and yellow in overlay. B) Quantitation of Mcoln1 WT and truncation mutant colocalization with EEA1 and Lamp1. Results are calculated from the extent of Mcoln1 signal within the region marked by EEA1 or Lamp1 expressed as a percent the total Mcoln1 signal in that cell. Values are means ± standard errors of ≥40 cells for each bar. C) Colocalization of Mcoln1 C-terminal truncation mutants P527* and Y521* with endogenous GM130, a marker for the Golgi apparatus. Bars, 10 μm. D) Quantitation (as described in B) of Mcoln1 WT and P527* and Y521* mutant colocalization with the Golgi marker, GM130. Values (% colocalization) are means ± standard errors of ≥30 cells for each bar.

The truncation studies described above utilized HSFs because these flat cells allow for easy morphological discrimination between different cellular compartments such as early endosomes and lysosomes. However, for examination of the extent of Mcoln1 fragmentation by immunoblotting, we expressed the Mcoln1 C-terminal truncations in HeLa cells because lysates of HSFs yielded insufficient Mcoln1 protein for such studies. When expressed in HeLa cells, immunoblotting showed that the series of C-terminal truncation mutants exhibited a progressive decrease in degradation compared to wild type Mcoln1 (Fig. 4). The extent of Mcoln1 fragmentation (Fig. 4B) in the truncation mutants was positively correlated (r²=0.99) with their extent of association with Lamp1 (Fig. 3C). These data suggest that Mcoln cleavage can be used as a tool to assess lysosomal delivery of Mcoln1.

Fig. 4. Reduced Mcoln1 cleavage in C-terminal truncation mutants.

HeLa cells were transfected with WT Mcoln1 or C-terminally truncated mutants described in Fig. 2. After 48 h, cell lysates were collected and analyzed by immunoblotting with the ML1-N antibody. A) Example of an immunoblot showing successively reduced fragmentation (decreased ~35 kDa band) with increased Mcoln1 C-terminal truncation. B) Quantitation of extent of fragmentation of Mcoln1 and C-terminal mutants. Blots were scanned and analyzed by image analysis. Percent fragmentation was calculated as 100 × intensity of the ~35 kDa fragment band/intensity of the fragment + intact Mcoln1. Results are means ± standard error for 3 blots.

We attempted to identify specific regions within the C-terminus of Mcoln1 that were responsible for efficient Golgi exit. Thus, we mutated individual residues to alanine in the region of amino acids 521-539 of Mcoln1. However, none of these point mutations caused a significant alteration in Mcoln1 targeting (Suppl. Fig. 2A). Notably, mutation to alanine of tyrosine 521, the first amino acid of a putative YXXØ motif, had no effect on Mcoln1 distribution (Suppl. Fig. 2A). Deletion of the entire region (del521-539) of Mcoln1 caused a slight decrease in targeting to lysosomes and a small decrease in degradation (Suppl. Fig. 2), but did not replicate the effects of the C-terminal truncations, P527* or Y521*. We conclude that the 521-539 region of Mcoln1 may provide secondary structure or spacing, rather than a specific amino acid motif, which is important for efficient exit of Mcoln1 from the Golgi apparatus.

As noted in the Introduction, depletion of AP1 using siRNA was reported to inhibit the exit of Mcoln1 from the Golgi apparatus and its delivery to lysosomes (25). We attempted to replicate this work to investigate the effects of AP1 on Mcoln1 fragmentation. HeLa cells were depleted of AP1γ >90 % using siRNA (Fig. 5A). However. AP1 depletion had no impact on the lysosomal delivery of transfected Mcoln1 as assessed by a lack of effect on Mcoln1 fragmentation (Fig. 5A0 or Mcoln1 colocalization with endogenously expressed CD63 (Fig. 5B,C). Thus, we were unable to find any evidence that full length Mcoln1 trafficking was dependent upon AP1. We also found that Mcoln1 lysosomal localization was not altered in cells deficient in AP3 (Suppl. Fig. 3A,B), as previously reported (25), or in cells in which clathrin-mediated endocytosis was disrupted by EPS15 mutant expression (Suppl. Fig. 3C).

Figure 5. Lack of effect of AP1 knockdown on Mcoln1 targeting.

HeLa cells were treated with 2 rounds of siRNA against AP1γ (or mock treated in the case of controls) over 6 days. During the second treatment, cells were also transfected with Mcoln1 (or indicated mutants) for 16 h. Cells were then lysed for immunoblotting or processed for immunofluorescence. A) Immunoblots showing expression of AP1-γ and Mcoln1. Note lack of detectable AP1γ in samples treated with AP1γ siRNA (AP1-KD) compared to controls. Detection of Mcoln1 (right panel) shows that Mcoln1 was significantly degraded to the ~35 kDa fragment, even when AP1γ was depleted. B) Colocalization by immunofluorescence of Mcoln1 WT with CD63 ± AP1γ-KD. Note extensive colocalization (yellow color) of Mcoln1 and CD63 in overlay images at right, even with AP1γ KD. Bar, 10 μm. C) Quantitation of Mcoln1 colocalization with Lamp1 ± AP1γ knockdown. Values are means ± standard errors of ≥40 cells from 2 independent experiments.

Inhibition of Mcoln1 Golgi exit and lysosomal delivery by kinase-inactive PKDs

Finally, we investigated the possibility that Mcoln1 trafficking was regulated by PKDs. PKDs have been shown to be involved in the Golgi exit of a number of plasma membrane proteins by regulating the fission of transport vesicles at the trans-Golgi network (8, 9); however, the role of PKDs in trafficking of lysosomal proteins is unknown. Thus, we co-expressed Mcoln1 and GFP-labeled, kinase-inactive forms of PKD1 and PKD2 (GFP-PKD1-KI and GFP-PKD2-KI) in HeLa cells. Both of these PKD proteins were distributed mainly in perinuclear structures resembling the Golgi complex (Fig. 6A). Such PKD-KI-positive compartments have been previously demonstrated to be highly tubulated, trans-Golgi network-derived structures (8, 9). In cells expressing GFP-PKD1-KI or GFP-PKD2-KI, Mcoln1 distribution was altered such that it was often visualized associated with the GFP-PKD1-KI-positive compartment (FIg. 6A). In addition, the extent of Mcoln1 localization with lysosomes was decreased in cells expressing GFP-PKD1-KI or GFP-PKD2-KI, as shown by decreased colocalization between Mcoln1 and the lysosomal markers, CD63 and Lamp1 (Fig. 6A,B). The distribution of Mcoln1 co-expressed with GFP-PKD-KIs for 16 or 24 h (data not shown) was similar to the 20 h transfection results shown in Fig. 6.

Figure 6. Over-expression of kinase inactive (KI) PKD inhibits Mcoln1 delivery to the lysosomes.

HeLa cells were transfected with Mcoln1 plus either GFP (control), GFP-PKD1-KI, or GFP-PKD2-KI for 20 h. Cells were fixed and immunostained for Mcoln1 and either endogenous CD63 or Lamp1. A) Representative Images are shown for green (GFP or GFP-PKD-KI), red (CD63) and far red (Mcoln1) flourescence in transfected cells. For clarity, images are pseudocolored with GFP in blue, CD63 in red and Mcoln1 in green. Overlay of control (top right) does not include GFP channel. Note extensive colocalization (yellow) of Mcoln1 and CD63 in control overlay (top right). In cells transfected with PDK1 or PKD2, Mcoln1 is little colocalized with CD63 (note separation of red and green signal in middle right and lower right panels) and instead Mcoln1 is distributed in a perinuclear mass (see arrows) which overlaps with the PKD-positive compartment. Bar, 10 μ. B) HeLa cells were transfected with Mcoln1 plus GFP-PKD1-KI, GFP-PKD2-KI or without a second DNA construct (controls) for 20 h and processed for immunofluorescence as above. Extent of colocalization between Mcoln1 and CD63 or Lamp1 was determined as in Fig. 3. Results are means ± SE from ≥ 100 cells from 3 separate transfections per condition. * indicates significant differences (p<0.001) in two tailed T-tests relative to corresponding controls.

As demonstrated by immunoblotting, the extent of Mcoln1 fragmentation was delayed when Mcoln1 was co-expressed with PKD isoforms (Fig. 7). Representative immunoblots show an increased ratio of intact vs. cleaved Mcoln1 at 16 and 20 h in cells co-expressing GFP-PKD2-KI compared to cells expressing GFP only (controls) (Fig. 7A). Quantitations of immunoblots demonstrated a significant inhibition of degradation (30–40%) occurring 20 h after transfection of HeLa cells in cells expressing GFP-PKD1-KI or GFP-PKD2-KI compared to controls (Fig. 7B). Together, these studies suggest that disruption of PKD function inhibits the exit of Mcoln1 from the Golgi, thus delaying the delivery of Mcoln1 to the lysosomes.

Figure 7. Over-expression of PKD-KI delays Mcoln1 degradation.

HeLa cells were transfected with Mcoln1 plus either GFP (control), GFP-PKD1-KI, or GFP-PKD2-KI for 16, 20 or 24 h as indicated. Cell lysates were prepared and blotted for Mcoln1 using antibodies that recognize the N- (ML1-N) and C-(ML1-C) termini of the protein. A) Representative western blots showing effect of PKD2-KI on Mcoln1 degradation at 16 and 20 h. Note the increased intact ~65 kDa Mcoln1 and/or decreased ~35 kDa fragment in PKD2-KI-expressing cells detected with both the ML1-C and ML1-N. B) Quantitation of Mcoln1 degradation at different time points ± PKD1-KI or PKD2-KI. Percent degradation was calculated as described in Fig. 4. Results are means ± SE from ≥ 3 samples per condition. * indicates values that were significantly different (p<0.05) than controls at the same time point in 2-tailed t-tests. Note the reduced degradation of Mcoln1 in PKD-KI expressing samples at the 20 h time point, compared to controls.

Since PKDs are known to be involved in the delivery of some PM proteins (8), it was of interest to determine if the expression of kinase-inactive PKD affected the transport of Mcoln1 mutants that lack N-and C-terminal dileucine motifs (L15-16A/L577-578A and L15-16A/G558*) and accumulate primarily at the PM (see Suppl. Fig. 1). WT, L15-16A/L577-578A and L15-16A/G558* forms of Mcoln1 were coexpressed in HeLa cells along with either GFP (controls) or GFP-PKD2-KI. GFP-PKD2-KI did not prevent the transport of these Mcoln1 mutants to the PM, although a portion of the Mcoln1 mutants could be observed in the GFP-PKD2-KI-positive compartment (Suppl. Fig. 4A). To quantitate PM deliver, after transfection, PM proteins were biotinylated and isolated using strepavidin-agarose beads.. Significant levels of both Mcoln1 mutants were detected associated with streptavidin beads (i.e., biotinylated at the PM); however, GFP-PKD2-KI had no significant effect on the PM levels of the Mcoln1 mutants at either 20 or 40 h after transfection (Suppl. Fig. 4B,C). Biotinylation studies of cells transfected with WT Mcoln1 showed no detectable Mcoln1 at the PM (Suppl. Fig. 4B), supporting the idea that little or no WT Mcoln1 is delivered to the PM while en route to the lysosomes.

DISCUSSION

We studied the Golgi exit and lysosomal transport of Mcoln1 by transfection of untagged Mcoln1 to minimize alterations (e.g., fluorescent protein or epitope tags) in the protein that might affect trafficking. Using a series of C-terminal truncations, we identified a region of the C-terminus of Mcoln1 (amino acids 540-580) as important for efficient targeting of this protein. Mutants lacking this 53 amino acid C-terminal region exhibited reduced delivery to the lysosomes and were retained partially in the Golgi apparatus, as revealed by immunofluorescence studies. We were unable to inhibit Mcoln1 transport by depletion of AP1. However, we were able to demonstrate that Mcoln1 delivery from the Golgi to lysosomes is inhibited by PKD-KI expression. We used immunoblotting to investigate the proteolytic cleavage of Mcoln1 under various conditions. Removal of dileucine motifs, which causes accumulation of Mcoln1 at the PM, as well as C-terminal truncation and PKD-KI expression, which cause retention of Mcoln1 in early secretory compartments, each significantly reduced Mcoln1 fragmentation. These results support the idea that Mcoln1 fragmentation requires the transport of Mcoln1 to lysosomes. We conclude that Mcoln1 is normally transported from the Golgi by a PKD-dependent mechanism and that transport to the lysosomes is necessary for Mcoln1 cleavage.

We utilized immunoblotting to study the extent of Mcoln1 fragmentation under various conditions. We showed that Mcoln1 mutants which lack both N- and C-terminal dileucine motifs (L15-16A/L577-578A and L15-16A/G558*) were distributed mainly at the plasma membrane, exhibited reduced colocalization with lysosomal markers and had decreased fragmentation (Fig. 1D). In addition to the dileucine motifs, we found that a series of C-terminal truncations of Mcoln1 led to progressively reduced lysosomal delivery and increased retention in the Golgi apparatus (Fig. 2 and 3), and that reduced lysosomal presence was significantly correlated with decreased Mcoln1 fragmentation (Fig. 4). Finally, we found that PKD-KI expression inhibited the transport of Mcoln1 to lysosomes, caused retention of Mcoln1 in a PKD-positive compartment and inhibited Mcoln1 degradation (Fig. 6, 7). Together, these data strongly support the concept that Mcoln1 fragmentation requires its delivery to lysosomes, an idea originally expressed by Kiselyov et al. (26). The biological function of Mcoln1 fragmentation is unknown but it has been proposed to be a mechanism for rapid inactivation of the protein after reaching the lysosomes (26).

Several groups have shown that the N and C-terminal dileucine motifs are important for the targeting of Mcoln1. Removal of either the N or C-terminal motif does not inhibit Mcoln1 transport to lysosomes. However, in the absence of both dileucine regions, Mcoln1 is distributed mainly at the plasma membrane [(12, 24) and Suppl. Fig. 1]. Acidic dileucine motifs are reportedly involved in the recognition of proteins by adaptor protein complexes (e.g., AP1-3) and GGAs (3–5). Vergarajauregui and Puertollano (24) reported that Mcoln1 C-terminal dileucine motif was targeted to lysosomes by an AP-2-dependent mechanism after delivery to the plasma membrane, whereas the N-terminal motif was directed to lysosomes independently of AP-2. This group suggested that Mcoln1 was targeted to lysosomes by multiple mechanisms. In contrast, Miedel et al. (25) reported that the delivery of full length Mcoln1 to lysosomes was AP1-dependent. We were unable to inhibit Mcoln1 transport by AP1 knockdown, even though AP1 expression was reduced by >90%. The observation that Mcoln1 degradation in AP1-depleted cells was not inhibited compared to controls (Fig. 5A) supports the idea that Mcoln1 transport to lysosomes was undisturbed by AP1 knockdown. We also were unable to inhibit Mcoln1 transport to lysosomes by disrupting clathrin endocytosis via Eps15 mutant expression or by depleting AP3 (Suppl. Fig. 3). Although clathrin adaptors such as AP1 and AP-2 are suggested to be important for Mcoln1 transport by the requirement for dileucine motifs, our results suggest that in the event that a single such mechanism is blocked, another mechanism is available to sort and deliver Mcoln1. Thus, our studies support the idea that Mcoln1 transport occurs by multiple and redundant mechanisms. The recognition of Mcoln1 by redundant sorting mechanisms suggests that Mcoln1 transport to its correct location may be essential for cell activities.

PKDs have be shown to be important for the Golgi exit of several proteins destined for the plasma membrane (8, 9, 28). We found that co-expression of Mcoln1 with PKD-KI led to trapping of a large portion of the expressed Mcoln1 in the PKD-positive compartment and inhibited the targeting of Mcoln1 to lysosomes (Fig. 6). In addition, PKD-KI expression delayed the fragmentation of Mcoln1 (Fig. 7), supporting the idea that trafficking to lysosomes was inhibited. These data suggest that PKDs play a role in the targeting of Mcoln1. Both PKD1 and PKD2 kinase-dead mutants inhibited the transport of Mcoln1. HeLa cells are reported to express endogenous PKD2 and PKD3 but little or no endogenous PKD1 (7). Thus, the expression of PKD1-KI in Hela cells may act as a dominant negative by interacting with effectors for PKD2 or PKD3. Alternatively, since PKD isoforms have been shown to interact (7), it is possible that PKD1-KI may heterodimerize with endogenous PKD2 or PKD3 in HeLa cells and thereby inhibit their function.

To our knowledge, these studies represent the first example of the involvement of PKDs in the trafficking of a protein destined for lysosomes Our results raise the possibility that PKDs might play a role in the transport of other transmembrane lysosomal proteins besides Mcoln1. Preliminary experiments suggested that PKD-2-KI inhibits the lysosomal delivery of GFP-lgp120 and CD63-GFP (DLM, unpublished data). The finding that multiple lysosomal proteins are regulated by PKDs would be especially interesting in light of the multiple mechanisms and itineraries involved in the Golgi exit and transport of lysosomal proteins.

METHODS

Cells and Cell Culture

Normal human skin fibroblasts (HSFs) (Coriell Institute, Camden, NJ) were cultured in Eagle’s minimal essential medium with 10% fetal bovine serum. Hermansky-Pudlak, type 2 (HPS-2) fibroblasts were from William A. Gahl, NIH, and were cultured as above. HeLa cells (American Type Culture Collection; Rockville, MD) were cultured in Dulbecco’s minimal essential medium with 10% fetal bovine serum. Cells were cultured to ~30–50% confluency on 35 mm acid-etched glass cover slips for microscopy, or in 100-mm dishes for biochemical experiments. In one set of experiments, HeLa cells were treated with 10 μM leupeptin (Sigma) for 48 h prior to collection of cell lysates.

Transfections and siRNA treatments

Fibroblasts were transfected by electroporation using an Amaxa electroporation system (Lonza; Basel, Switzerland). Hela cells were transfected with 1 μg/ml plasmid DNA and 3μl/ml FuGENE 6 (Roche Applied Science, Indianapolis, IN) per ml of culture medium. Transfected cells were used for experiments after 16–48 h. For AP1 knockdown, Hela cells were treated with two rounds of 100 μM Smart Pool siRNA directed against human AP1γ (Invitrogen) over 6 days using Lipofectamine 2000 (Invitrogen), according to manufacturers instructions. Controls for these knock down experiments were subject to the same treatment but without siRNA.

Constructs

The full open reading frame of Mcoln1 cDNA wasamplified from normal humanskin fibroblast cDNA by RT-PCR using primers (5′-GGGCTAGCGAGCGAGGTCGCAGTGAC; 3′-GTAGATCTGCGACAGGGAGCCGATCC) that added Nhe I and Bgl II sites to the 5′ and 3′ ends, respectively. The PCR product was cloned into the NheI/BglII sites of the pIRES2-EGFPvector (BD Clontech, Palo Alto, CA) and sequenced. The Mcoln1 coding sequence in pIRES2-EGFP was amplified by RT-PCR (5′ primer as above; 3; primer-GGAAGCTTATTCACCAGCAGCGAATG) which added a Hind III site. Mcoln1 was then inserted into pDsRed2-N1 (Clontech, Mountain View CA) at the NheI and HindIII sites to create pMcoln1-DsRed2. Untagged Mcoln1 with no fluorescent reporter was prepared by introducing a termination codon at the 3′ end of the Mcoln coding sequence in pMcoln1-DsRed2, thus eliminating the DsRed tag. This point mutation, as well as other point mutations and truncations in Mcoln1 were introduced into the pMcoln1-DsRed2 construct using the Quikchange kit (Stratagene, La Jolla, CA) using appropriate primers, and verified by DNA sequencing using an ABI PRISM^™ 377 DNA Sequencer (Perkin-Elmer Applied Biosystems, Foster City, CA) in the Mayo Clinic Molecular Biology Core Facility.

pGFP-PKD1-KI in which lysine 618 of PKD1 is mutated to asparagine and pGFP-PKD2-KI in which lysine 580 of PKD1 is mutated to asparagine were gifts from Vivek Malhotra, Centre for Genomic Regulation, Barcelona Spain. GFP-labeled dominant negative Eps (15 EH1 mutant ED95/295) was from A. Benmerah and A. Dautry-Varsat (Institut Pasteur, Paris, France)

Mcoln1 Antibodies

Peptides with sequences TQAGPSPAPPTPPEEEDLRRRLK and EESELQAYIAQCQDSPTSGKFRRGSGS corresponding to regions near the N- and C-termini of Mcoln1, respectively, were synthesized (Mayo Proteomics Research Center, Rochester, MN) and used to immunize rabbits. The resulting antisera (ML1-N and ML1-C, against regions near the N and C-terminus of Mcoln1, respectively) were tested for their ability to recognize recombinant Mcoln1 expressed in HeLa cells and HSFs. For immunofluorescence studies, antibodies were purified using the corresponding peptide conjugated to AminoLink Plus columns (Thermo Scientific).

Immunoblotting

Hela cells transfected with Mcoln1 constructs were washed 3 times in ice cold phosphate buffered saline (10 mM NaH₂PO₄, 150 mM NaCl, pH 7.2; PBS), scraped from culture dishes in PBS and then centrifuged. The pelleted cells were then resuspended in 50 mM HEPES, 25 mM KCl, 20 % (v/v) glycerol with protease inhibitors (10 μg/ml each leupeptin and aprotinin, 1 μg/ml pepstatin, 25 μM 4-amidophenylmethansulfonyl fluoride, 1 mM phenylmethylsulfonyl fluoride), and homogenized by 6 passes through a 25 ga needle. Samples were dissolved in SDS-PAGE sample buffer with 7.5 mg/ml dithiothreitol, warmed to 37°C for 10 min, run on 10 % SDS-PAGE gels (29), and transferred to polyvinylidene difluoride membranes with 20% methanol transfer buffer as described (30). Membranes were blocked with 10% nonfat dry milk in PBS/3% Tween 20 (PBS-T), incubated with primary antibodies (1/1000) in 2% nonfat dry milk in PBS-T for 2 h at 37° C, and washed in PBS-T. Membranes were then incubated with peroxidase-conjugated secondary antibodies (Millipore, Billerica MA) in 2% nonfat dry milk in PBS-T for 1 h at room temperature. After washing membranes in PBS-T, protein bands were visualized on film using a chemiluminescence reagent (PerkinElmer Life Sciences, Boston, MA).

Cell Surface Biotinylation

The PM portion of Mcoln1 proteins expressed in HeLa cells was estimated by biotinylating surface-exposed proteins of living cells using EZ-Link Sulfo-NHS-SS-biotin reagent (Thermo) for 1 h at 4°C according to the manufacturer’s instructions. Cell samples were quenched with 25 mM Tris-HCl, 150 mM NaCl, pH 7.6, for 10 min and then lysed with RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) supplemented with Complete protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN) Biotinylated proteins were isolated by incubation with 100 μl streptavidin agarose beads (Thermo) at 4 °C for 1 h. The beads were washed with RIPA buffer and 2X SDS-PAGE sample buffer containing 40 mg/ml dithiothreitol was then added. Finally, Mcoln1 was detected by immunoblotting as above.

Immunofluorescence studies

For immunofluorescence studies, transfected cells on cover slips were fixed for 40 min in 3.2 % formaldehyde in PBS. After fixation the samples were permeabilized for 2 min with 0.1 % Triton X-100 in PBS, and blocked for 1 h with blocking buffer (10% normal goat serum, 5% glycerol, 0.5 % sodium azide in PBS). Cells were then co-incubated with Mcoln1 antibodies (~5 μg/ml) and monoclonal primary antibodies against early endosomal autoantigen (EEA1), CD63, calnexin (all from BD Biosciences, San Diego, CA), or Lamp1 (clone H4A3; Developmental Studies Hybridoma Bank, Iowa City, IA), in blocking buffer for 2 h at room temperature. After washing samples were incubated with AlexaFluor (488, 594 or 647) -conjugated secondary antibodies (Invitrogen) for 1 h. All samples were washed and then were mounted on slides in SlowFade Light (Invitrogen). In some studies Mcoln1 was co-expressed with PKD1-KI-GFP or PKD2-KI-GFP before processing for immunofluorescence. Fluorescence microscopy was carried out using an Olympus AX70 fluorescence microscope.

Data analysis

Quantitation of colocalization images was performed using the colocalization feature of the Metamorph imaging program (Universal Imaging Corp. Downington, PA) using the integrated method option. Colocalization results are expressed as mean (± standard error) percent of Mcoln1 signal overlapping with marker (i.e., 100 × (area × intensity of Mcoln1 overlap with marker)/(area × intensity of total Mcoln1 signal). Scanned images of immunoblots were analyzed quantitatively using ImageJ 1.42q (NIH). Statistical analyses were conducted by using T-test or correlation analysis features within Microsoft Excel.

Supplementary Material

Supp Fig S1-S4

Suppl. Fig. 1. Cellular distribution of dileucine motif mutants of Mcoln1. Mcoln1 mutants in which the C-terminal (L577-578A), N-terminal (L15-16A), or both (L15-16A/L577-578A) dileucine groups were replaced by alanine and expressed in HSFs. A mutant in which the N-terminal leucines were altered to alanine and the C-terminus was truncated at amino acid 557 (L15-16A/G558*) was also studied. Cells were processed for immunofluorescence using antibodies against Mcoln1 (red) and the lysosomal marker, Lamp1 (green). The merged red and green images are shown at right (overlays). Note the extensive colocalization between mutants (L15-16A) and (L577-578A) and Lamp1 which is simlar to the localization of wild type Mcoln1 (see Fig. 2). In contrast, mutants L15-16A/L577-578A and L15-16A/G558*, which lack both dileucine motifs, localized predominantly to the PM (e.g., at arrows). A portion of these two mutants could be seen to localize to intracellular vesicles which colocalized with Lamp1 (e.g., at arrowheads). Bar, 10 μm.

Suppl. Fig. 2. Mutation of the 521-540 amino acid region of Mcoln1. A) Amino acids in the 521-540 region of full length Mcoln1 were mutated to alanine as indicated in bold at left. The wild type sequence is shown at the top. In one mutation, amino acids 521-539 were deleted. At right, quantitation of colocalization between each mutant and Lamp1 and EEA1 are shown (see legend of Fig. 3 for details). Results are expressed as percent colocalization between Mcoln1 and markers and are means ± SE for ≥20 cells per mutant vs. marker. B) Extent of degradation of WT and del521-539 Mcoln1 as assessed by immunoblotting using the ML1-N antibody. C) Quantitation of degradation of WT and del521-539 Mcoln1 (as in Fig. 4B). Results are means ± SE of three blots.

Suppl. Fig. 3. Mcoln1 localization is independent of AP3 and clathrin-mediated endocytosis. A) Mcoln1 was transfected into normal HSFs or in HPS-2 mutant fibroblasts (deficient in β3A of the AP-3 complex) for 48 h and colocalized with Lamp1 by immunofluorescence. Mcoln1 signal (red) and Lamp1 signal (green) are overlayed at right. Bar, 10 μm. B) Mcoln1 was expressed in normal and HPS-2 fibroblasts as in A and the extent of Mcoln1 colocalization with Lamp1 and EEA1 was quantitited (as in Fig. 3). Results are means ± SE for ≥20 cells for each bar. C) Mcoln1 and GFP-EPS15-dominant negative (or GFP for controls) were co-transfected into HeLa cells. After 48 h, immunofluorescence for Mcoln1 and CD63 was performed and colocalization of Mcoln1 with CD63 was quantified. Values are means ± SE from <40 cells per condition from 2 separate experiments.

Suppl. Fig. 4. PM delivery of Mcoln1 mutants is not dependent on PKD2. HeLa cells were cotransfected with Mcoln1 mutants lacking dileucine motifs (L15-16A/L577-578A or L15-16A/G558*) or WT Mcoln1, and either GFP (controls) or GFP-PKD2-KI. A) Immunofluorescence showing the distribution of Mcoln1 mutants (upper panel) after cotransfection with GFP or GFP-PKD2-KI for 40 h. The corresponding GFP signals for the same fields are shown in the lower panels. Dotted lines indicate the perimeters for cells transfected with GFP-PKD2-KI. B and C) After transfection, live cells were treated with sulfo-NHS-SS-biotin at 4°C to biotinylate surface proteins. Cell samples were then lysed and biotinylated proteins were isolated using strepavidin-agarose beads. Samples of total and biotinylated proteins were then analyzed by immunoblotting for Mcoln1. B) Representative immunoblots showing total and PM levels for WT and mutant Mcoln1 proteins in lysates of cells transfected for 40 h. Total samples (initial lysates) were loaded at 20 μg each. PM samples (biotinylated fractions isolated on beads) were loaded at an equivalent to 3 times the protein loaded for total samples based on the amount of protein initially added to beads. C) Quantitation of PM/Total ratios of Mcoln1 mutants in lysates from cells transfected for 20 or 40 h. Ratios of PM/Total for the Mcoln1 mutant proteins were calculated from immunoblots as shown in (A). Results are means ± SE for ≥4 samples for each condition. Note the detection of Mcoln1 at the PM for the dileucine mutants. No significant differences in PM/Total ratios were detected for GFP-PKD2-KI vs. GFP treatments for either mutant at 20 or 40 h.