Manganese Redistribution by Calcium-stimulated Vesicle Trafficking Bypasses the Need for P-type ATPase Function*

Background: Yeast is a model system for the study of mechanisms governing eukaryotic Golgi-Mn2+ homeostasis. Results: We provide evidence that calcium stimulates ER and late endosome/trans- to cis-Golgi manganese delivery and bypasses the need for Pmr1. Conclusion: Vesicle trafficking promotes organelle-specific ion interchange and cytoplasmic metal detoxification. Significance: Our findings open new perspectives on chemical modifiers of Hailey-Hailey disease. Regulation of intracellular ion homeostasis is essential for eukaryotic cell physiology. An example is provided by loss of ATP2C1 function, which leads to skin ulceration, improper keratinocyte adhesion, and cancer formation in Hailey-Hailey patients. The yeast ATP2C1 orthologue PMR1 codes for a Mn2+/Ca2+ transporter that is crucial for cis-Golgi manganese supply. Here, we present evidence that calcium overcomes the lack of Pmr1 through vesicle trafficking-stimulated manganese delivery and requires the endoplasmic reticulum Mn2+ transporter Spf1 and the late endosome/trans-Golgi Nramp metal transporter Smf2. Smf2 co-localizes with the putative Mn2+ transporter Atx2, and ATX2 overexpression counteracts the beneficial impact of calcium treatment. Our findings suggest that vesicle trafficking promotes organelle-specific ion interchange and cytoplasmic metal detoxification independent of calcineurin signaling or metal transporter re-localization. Our study identifies an alternative mode for cis-Golgi manganese supply in yeast and provides new perspectives for Hailey-Hailey disease treatment.

The intracellular levels of ions and other micronutrients are closely regulated in eukaryotic cells. This is the case for the trace element manganese (Mn 2ϩ ), whose regulation is particularly important. This redox active metal is a key cofactor for a wide range of enzymes located in every cellular compartment (1). However, at high concentrations Mn 2ϩ is toxic and promotes DNA damage coupled to replication defects in yeast (2). In humans, overexposure to Mn 2ϩ results in a neurological syn-drome called manganism, whose symptoms resemble those of Parkinson disease (3). In addition, Mn 2ϩ has been shown to favor prion misfolding if it displaces copper as the protein cofactor (4). Hailey-Hailey disease phenotypes have been associated with mutations affecting calcium and/or manganese transport activities of the Golgi Ca 2ϩ /Mn 2ϩ transporter ATP2C1 (5). A representative Hailey-Hailey phenotype caused by alterations in the intracellular Mn 2ϩ flux includes keratinocyte differentiation (6). For these reasons, revealing the intracellular mechanisms that regulate Mn 2ϩ homeostasis pathways is of clinical importance.
Much of our current understanding of eukaryotic manganese homeostatic mechanisms comes from the budding yeast, Saccharomyces cerevisiae. Yeast Mn 2ϩ uptake is provided by the plasma membrane transporter Smf1, a member of the natural resistance-associated macrophage protein (Nramp) 4 family (7). Smf2 represents a member of intracellular Nramp Mn 2ϩ transporters essential for the activity of Mn 2ϩ -dependent enzymes, which include the mitochondrial Sod2 protein and Golgihosted sugar transferases (8). Smf2 localizes to Golgi-like vesicles, and a drop in whole-cell Mn 2ϩ has been observed upon SMF2 deletion (8). Under physiological conditions, ϳ90% of newly synthesized Smf1 and Smf2 are directly targeted to the vacuole for degradation, presumably to limit uptake of toxic Mn 2ϩ amounts (9,10). When Mn 2ϩ becomes limiting, these transporters are delivered to the cell surface (Smf1) and intracellular vesicles (Smf2) to increase Mn 2ϩ uptake (9,10). In contrast, in conditions of toxic metal concentrations, the vacuolar degradation of the Nramp transporters is enhanced, and Smf1 is virtually eliminated from the plasma membrane (11). Moreover, Mn 2ϩ uptake by manganese-phosphate complexes is facilitated by the high affinity cell surface phosphate transporter Pho84 (12).
Other factors that influence intracellular Mn 2ϩ homeostasis include the putative Mn 2ϩ transporter Atx2. Atx2 localizes to Golgi-like vesicles, but the mechanism by which Atx2 regulates intracellular Mn 2ϩ levels remains unknown (13). Recently, the P-type ATPase Spf1 (hATP13A1) has been suggested to regulate Mn 2ϩ transport into the endoplasmic reticulum (ER) (14), whereas Pmr1, a Golgi-localized P-type Ca 2ϩ and Mn 2ϩ ATPase, pumps cytosolic Mn 2ϩ into the lumen of the Golgi (15)(16)(17). Apart from providing sugar transferases with Mn 2ϩ as cofactor, Pmr1 has another role in Mn 2ϩ detoxification by secretory pathway-mediated excretion (16 -18). In addition to Pmr1, Mn 2ϩ detoxification can be carried out by the vacuolar iron and manganese transporter Ccc1 (19).
Membrane fission and fusion are essential processes, allowing the dynamic communication between membrane-bounded organelles in all eukaryotic cells. Lipid vesicles are constantly emerging from one membrane to fuse with another, providing transport shuttles between distinct intracellular compartments. Increasing evidence suggests that calcium (Ca 2ϩ ) plays a role in the regulation of membrane trafficking. For example, Ca 2ϩ appears to be involved in ER to Golgi transport (20), intra-Golgi transport (21), and early endosome fusion (22) as well as yeast homotypic vacuole fusion (23).
Although many players involved in the intracellular manganese trafficking network have been characterized in yeast, our understanding of organelle-to-organelle Mn 2ϩ flux is far from complete. Here, we report a Pmr1-independent mechanism for cis-Golgi Mn 2ϩ supply. This supply depends on the ER Mn 2ϩ transporter Spf1 and the Smf2 late endosome/trans-Golgi Mn 2ϩ transport activity and can be counteracted by ATX2 overexpression. In addition, it requires extracellular CaCl 2 in order to stimulate vesicle trafficking and membrane fusion. Based on our observations we propose a model on intracellular manganese homeostasis that provides mechanisms for intraorganelle ion flux and manganese detoxification.

EXPERIMENTAL PROCEDURES
Yeast Strains and Plasmids-Yeast strains and plasmids used in this study are listed in Table 1. Gene deletions were constructed by PCR-based methods using pAG25 (EUROSCARF) and pFA6a-klLEU2MX6 (kindly provided by B. Pardo) as template plasmids. In other cases strains were derived from genetic crosses. The chromosomal SMF2 open reading frame under the control of its own promoter was C-terminal-tagged with enhanced GFP (eGFP) by a PCR-based method using the tagging vector pKT209 (pFA6a-link-yEGFP-CaURA3) (24) as the template plasmid. To generate plasmid pNG011, SMF2 was amplified from genomic DNA, digested with EcoRI/SalI, and inserted into EcoRI/SalI site of pUG23 (25). To generate plasmid pNG026, ATX2 and mCherry were amplified from genomic DNA or pKS39 (26), respectively, using overlapping oligonucleotides. The PCR products were mixed and amplified using external oligonucleotides, digested with BamHI/Sac1, and inserted into BamHI/Sac1 site of p2UGpd (27).
Drug Sensitivity Assays-Yeast cells were adjusted in concentration to an initial A 600 of 0.2, then serially diluted 1:10 and spotted onto plates without or with different drugs at the indicated concentrations (see figure legends). CaCl 2 was added when indicated. Plates were then incubated at 30°C for 3-4 pUG23, METp-SMF2-GFP This study pNG026 p2UGpd, GPDp-ATX2-mCherry This study pRS315-HA-GFP-cSNC1 CEN, LEU2, HA-GFP-cSNC1 (53) days, except for temperature-sensitive mutants, which were incubated at the corresponding permissive or semipermissive temperatures.
Pulse-Chase Analysis of CPY-Pulse-chase labeling and analysis of immunoprecipitates was done as described previously (28).
Analysis of Telomere Length-Genomic DNA was isolated from yeast strains grown in YPAD for 3 days with or without the addition of 10 mM CaCl 2 . DNA was digested with XhoI, separated on a 1% agarose-Tris borate EDTA gel, transferred to a Hybond XL (Amersham Biosciences) membrane, and hybridized with a 32 P-labeled DNA probe specific for the terminal YЈ telomere fragment. The probe was generated by random hexanucleotide-primed DNA synthesis using a short YЈ specific DNA template, which was generated by PCR from genomic yeast DNA using the primers YЈ up (5Ј-TGCCGTGCAACAA-ACACTAAATCAA-3Ј) and YЈ low (5Ј-CGCTCGAGAAAGT-TGGAGTTTTTCA-3Ј). Three independent colonies of each strain were analyzed to ensure reproducibility.
Fluorescence Microscopy-Plasmid harboring yeast cells were grown to mid-log-phase in selective Synthetic Complete (SC) medium to maintain the plasmid and fixed in 2.5% formaldehyde and 0.1 M potassium phosphate buffer, pH 6.4, for 10 min. Cells were then washed twice with 0.1 M potassium phosphate buffer, pH 6.6, and finally resuspended in 0.1 M potassium phos-phate buffer, pH 7.4. Cells were imaged at 25°C using a microscope (DM-6000B, Leica) at 100ϫ magnification using L5, N3, and TX2 filters and a digital charge-coupled device camera (DFC350, Leica). Images were taken using LAS AF software (Leica) with the same exposure times for Smf2-GFP (1s) and lower exposure times for different marker proteins in the colocalization analysis. Images were assembled in Photoshop (Adobe) with only linear adjustments. Statistical analysis of colocalization was performed by counting at least 100 cells per marker derived from three independent experiments. Data are shown as the mean Ϯ S.D.
Metal Measurements-Yeast cells were grown to an A 600 of 2.5 in YPAD medium or the same medium supplemented with 5 mM CaCl 2 . In both cases the growth media was supplemented with 20 M MnCl 2 to monitor metal accumulation under manganese toxicity conditions. The cultures were harvested and washed with TE (10 mM Tris-HCl and 1 mM EDTA, pH 8), then deionized water, and finally dried. Samples were subjected to acid digestion and applied to an ICP Horiba Jobin Yvon Ultima 2 atomic-emission spectrometer at the Microanalysis Service of University of Seville (Seville, Spain). Manganese and calcium content were measured according to the manufacturer's specifications.
Microarray Analysis-Gene expression profiles were determined by using the "3Ј-expression microarray" technology by Accumulation of manganese (Mn) and calcium (Ca) in WT and pmr1⌬ cells without or with the addition of CaCl 2 (10 mM) was determined by inductively coupled plasma atomic emission spectrometry as described under "Experimental Procedures." Error bars represent S.D. B, CaCl 2 restores WT telomere length in pmr1⌬ mutants. WT and pmr1⌬ cells were grown in YPAD without or with the addition of 10 mM CaCl 2 for 3 days. Genomic DNA was isolated from the strains, digested with XhoI, and subjected to Southern blot (see "Experimental Procedures"). The location of the terminal YЈ telomere fragments is indicated. The dashed white line marks the telomere size of WT. C, extracellular CaCl 2 bypasses pmr1⌬ smf1⌬ lethality. Shown is tetrad analysis crossing pmr1⌬ with smf1⌬ without (left) or with (right) 10 mM CaCl 2 in the medium. The genotype of the relevant spores is indicated.

Intra-Golgi Manganese Redistribution
Affymetrix platform at the Genomics Unit of CABIMER (Seville, Spain) as described previously (2), with the modification that total RNA was isolated from cultures grown on YPAD ϩ 5 mM CaCl 2 .

CaCl 2 Counteracts Mn 2ϩ
Toxicity-In a previous work we found that an excess of cytosolic Mn 2ϩ alters mRNA transcription regulation and challenges genome stability (2). An example is the transcriptional 42-fold down-regulation of the low-affinity plasma membrane Mn 2ϩ transporter PHO84 (YML123C). Interestingly, upon CaCl 2 addition, transcriptional down-regulation of PHO84 was reversed, suggesting that extracellular CaCl 2 alters cellular Mn 2ϩ levels (see the supplemental data). To test if this is the case, we first compared the total cellular manganese and calcium levels in wild type and pmr1⌬ cells in the presence of extracellular CaCl 2 (Fig. 1A). In accordance with previous studies (16), pmr1⌬ cells suffered from a dramatic increase in total manganese and calcium levels. Upon the addition of CaCl 2 , the cellular calcium content increased with a concurrent decrease in the manganese content (ϳ8.5-fold). Because Mn 2ϩ interferes with telomerase activity leading to telomere shortening (29) we assayed telomere length variation as an indirect measure for nuclear Mn 2ϩ levels (Fig. 1B). We found that telomere shortening in pmr1⌬ mutants was alleviated upon CaCl 2 addition, suggesting that the addition of extracellular CaCl 2 either competes with Mn 2ϩ uptake or stimulates the removal of toxic Mn 2ϩ from the cytoplasm.
Transformation with an SMF1 overexpression vector challenged pmr1⌬ viability independently of CaCl 2 supplementation (data not shown), indicating that increased Smf1 levels could lead to uncontrolled and toxic Mn 2ϩ uptake. Loss of the Mn 2ϩ importer Smf1 should, therefore, impair Mn 2ϩ uptake and suppress pmr1⌬ phenotypes related to cytosolic Mn 2ϩ excess. However, deletion of SMF1 has been shown to be lethal in combination with pmr1⌬ (30) (Fig. 1C, left), whereas mutations in PMR1 up-regulate Smf1 protein levels under Mn 2ϩ starvation conditions (11). Interestingly, we could recover viable pmr1⌬ smf1⌬ spores when the tetrads were plated on CaCl 2 -containing medium (Fig. 1C, right), suggesting that CaCl 2 is able to facilitate bypass of Mn 2ϩ toxicity via an alternative mechanism.
Bypass of pmr1⌬ Glycosylation Defects Requires the Putative Mn 2ϩ Transporters Spf1 and Smf2-Numerous studies have reported suppression of other pmr1⌬ phenotypes by CaCl 2 (31)(32)(33). However, the underlying mechanism by which this occurs remains unclear. We asked whether other cation transporters contribute to this phenomenon. First, we set up a tar-geted, genetic screen for synthetic phenotypes of pmr1⌬ with deletion of genes involved in Ca 2ϩ or Mn 2ϩ homeostasis. As a read-out, we monitored pmr1⌬-dependent loss-of-viability by the cell wall-perturbing agent calcofluor white (CFW) (34,35) and recovery-of-viability in the presence of CaCl 2 . Consistent with glycosylation defects, pmr1⌬ shows a weakened cell wall exemplified by hypersensitivity to CFW, Congo Red, and hygromycin B and constitutive activation of the cell integrity pathway (36). Notably, CaCl 2 -mediated recovery of viability was not observed in other mutants affected in protein glycosylation such as anp1⌬, lacking a cis-Golgi ␣-1,6-mannosyltransferase subunit (Fig. 2A). Interestingly, CFW sensitivity of pmr1⌬ pho84⌬, pmr1⌬ vcx1, and pmr1⌬ ccc1⌬ double mutants was suppressed by CaCl 2 , whereas pmr1⌬ spf1⌬ and pmr1⌬ smf2⌬ double mutants failed to grow upon CaCl 2 addition (Fig.  2B).
Mn 2ϩ ions are essential cofactors for the activity of Golgihosted mannosyltransferases that progressively and sequentially N-glycosylate proteins in different Golgi compartments (18,37). Glycosylation events along the secretory route can be followed by analyzing carboxypeptidase Y (CPY) maturation. CPY is subjected to core glycosylation in the ER (p1 form). The core oligosaccharides are extended in the Golgi by the sequential addition of ␣1,6-, ␣1,2-, and ␣1,3-linked mannose residues, which results in a mobility shift when analyzed by SDS-PAGE (p2 form). After delivery to the vacuole, the pro region is cleaved to yield mCPY (see Fig. 2C, left) (38). In accordance with a previous report (31), fully glycosylated CPY (p2) was nearly absent in pmr1⌬ mutants, but CPY glycosylation recovered upon CaCl 2 addition. We confirmed the previously described CPY glycosylation defect of pmr1⌬ spf1⌬ double mutants (39), but surprisingly CPY glycosylation was significantly diminished in smf2⌬ mutants, and even more interestingly, we observed a CaCl 2 persistent glycosylation defect in smf2⌬, spf1⌬ single and pmr1⌬ smf2⌬, pmr1⌬ spf1⌬ double mutants. To further define the protein glycosylation defect of smf2⌬ mutants, we compared CPY mannosylation patterns by pulse-chase labeling and sequential immunoprecipitation with antibodies specific to either CPY or ␣1,6-mannose linkages (Fig. 2D). In contrast to pmr1⌬, smf2⌬ isolated CPY can be ␣1,6-mannosyl-immunoprecipitated, indicating a proficient early (cis-Golgi) ␣1,6-mannosyl addition. We, therefore, searched for evidence that the N-glycosylation defect of smf2⌬ cells might be linked to a late glycosylation event. Consequently, we assessed the subcellular localization of Smf2 by colocalization experiments with protein markers for the trans- does not suppress the CFW sensitivity of mutants lacking the cis-Golgi ␣-1,6-mannosytransferase complex subunit Anp1. WT, pmr1⌬, and anp1⌬ cells were grown to mid-log phase, serially diluted, and spotted onto YPAD or YPAD ϩ CFW (15 g/ml) without or with the addition of 10 mM CaCl 2 in the medium. Pictures were taken after 3 days. B, CaCl 2 fails to rescue CFW resistance of pmr1⌬ mutants in the absence of Spf1 or Smf2. Shown is CFW sensitivity of pmr1⌬ upon additional deletion of the low affinity Mn 2ϩ transporter PHO84 (12), the vesicular Mn 2ϩ transporter SMF2 (10), the vacuolar Ca 2ϩ /H ϩ exchanger VCX1 (68), the plasma membrane Ca 2ϩ channel CCH1 (69), the vacuolar Fe 2ϩ /Mn 2ϩ transporter CCC1 (19), and the putative ER Mn 2ϩ -transporter SPF1 (14). Drop test analysis of WT, pmr1⌬, pho84⌬, pmr1⌬ pho84⌬, smf2⌬, pmr1⌬ smf2⌬, vcx1⌬, pmr1⌬ vcx1, cch1, pmr1⌬ cch1⌬, ccc1⌬, pmr1⌬ccc1⌬, spf1⌬, and pmr1⌬ spf1⌬ cells is shown. See panel A for growth conditions. C, CaCl 2 failed to restore CPY glycosylation of pmr1⌬ mutants in the absence of Spf1 or Smf2. A schematic representation of CPY maturation is shown (left). Pulse-chase analysis of CPY maturation with or without the addition of 10 mM CaCl 2 is shown (right). Proliferating cells were radiolabeled for 5 min, chased for the indicated times, and lysed. CPY was immunoprecipitated, resolved by SDS-PAGE, and analyzed by phosphorimaging. ER (p1), Golgi (p2), and vacuole (m) CPY forms are indicated. D, smf2⌬ mutant is proficient in the ␣1,6-mannosyl addition. Cells were radiolabeled for 5 min and chased for 30 min. CPY was recovered by immunoprecipitation, split into two equal aliquots, subjected to secondary immunoprecipitation with antiserum to CPY or ␣1,6-mannose linkages, resolved by SDS-PAGE, and subjected to phosphorimaging analysis. Ab, antibody.
Smf2 and Atx2 Have Antagonistic Roles in Late Endosome/ Trans-Golgi Mn 2ϩ Transport-Another option would be that the late endosome/trans-Golgi could act as a cellular Mn 2ϩ storage compartment as previously proposed by Luk and Culotta (8). If so, we reasoned that a Mn 2ϩ exporter system might be required to prevent trans/post-Golgi Mn 2ϩ overload. A candidate for such activity is Atx2, based on the observations that Atx2 is a Golgi membrane protein whose overproduction provides the cytoplasm with antioxidative Mn 2ϩ activities that compensate for the loss of cytoplasmic SOD1, although Atx2 effect seems to require Smf1 function (13). To validate our hypothesis, we determined if ATX2 overexpression counteracts the CaCl 2 -mediated pmr1⌬ smf1⌬ viability (Fig. 4A). In fact, transformation of pmr1⌬ smf1⌬ double mutants with an ATX2 overexpressing plasmid conferred lethality in the presence of CaCl 2 , indicating a Smf1-independent function of Atx2. In addition, ATX2 overexpression compromised the CaCl 2 -dependent suppression of CFW sensitivity in pmr1⌬ mutants (Fig. 4B). These observations suggest that Atx2 might expel Mn 2ϩ from the trans-Golgi but also that enough Mn 2ϩ is available for Atx2-mediated Mn 2ϩ transport in CaCl 2 -treated pmr1⌬ smf1⌬ cells. We, therefore, determined if Atx2 and Smf2 co-localize to the same compartment (Fig. 4C). This was indeed the case, and based on our experimental evidence we anticipate that Smf2 and Atx2 might have antagonistic roles in trans-Golgi Mn 2ϩ homeostasis such that Smf2 and Atx2 are required for trans-/post-Golgi Mn 2ϩ import and export, respectively.

CaCl 2 -dependent Suppression Does Not Rely on Calcineurinmediated Signaling or Smf2 Redistribution from Trans-to
Cis-Golgi-Extracellular Ca 2ϩ has been shown to initiate signal transduction events (40). The conserved Ca 2ϩ /calmodulin-dependent protein phosphatase calcineurin plays a critical role in Ca 2ϩ -mediated signaling (41). Therefore, we scored CFW sensitivity of pmr1⌬ mutants compromised in the calcineurin regulatory subunit CNB1 or added calcineurin inhibitors (FK506 or cyclosporin A (CsA)) to the growth media (41) (see Fig. 5A). Neither lack of Cnb1 nor the addition of calcineurin inhibitors caused a loss-of-viability in the presence of CFW, suggesting that activation of calcineurin signaling is dispensable for CaCl 2 -mediated suppression of pmr1⌬ CFW hypersensitivity.
Smf2 could have a dual role in late endosome/trans-and cis-Golgi Mn 2ϩ import if one considers a CaCl 2 -dependent late endosome/trans-to cis-Golgi Smf2 redistribution. We addressed this possibility by determining the Smf2 subcellular localization in the presence of CaCl 2 and found that Smf2 still co-localized with the trans-Golgi marker Sec7 but not with the cis-Golgi marker Sed5 (Fig. 5B). Thus, the CaCl 2 -mediated suppression of cis-Golgi Mn 2ϩ import defect in pmr1⌬ does not occur through Smf2-mediated Mn 2ϩ redistribution from trans-to the cis-Golgi.

Rescue of pmr1⌬ CFW Resistance Relies on a Competent Golgi
Retrograde Transport Machinery-In addition to its function in cellular signaling, intracellular Ca 2ϩ also plays a regulatory role in membrane trafficking. In particular, Ca 2ϩ is thought to participate in different membrane fusion events within secretory and endocytic pathways including intra-Golgi transport (42) (see Fig. 6A). To determine if this is the case, we investigated whether intracellular transport and membrane fusion are essential for CaCl 2 -dependent suppression of glycosylation defects. First, we benefited from a sec18 -20 mutation that has been shown to block many vesicular fusion events (43,44). Sec18 is an essential ATPase that catalyzes the disassembly and recycling of SNARE complexes for further rounds of vesicle transport (45). Indeed, CaCl 2 failed to rescue growth of pmr1⌬ sec18 -20 double mutants on CFW-containing media, suggesting that vesicle transport is involved in CaCl 2 -dependent resistance to CFW (Fig. 6B). Next, to broadly assess vesicle trafficking steps, we took advantage of monensin, a Na ϩ /H ϩ ionophore that interferes with intracellular transport by the neutralization of acidic intracellular compartments (46), block-ing intracellular transport in both trans-and post-Golgi compartments (47). Most appealing, CaCl 2 failed to rescue the CFW resistance in the presence of monensin (Fig. 6C, left). We then considered that monensin constrains protein glycosylation in pmr1⌬ mutants. This was indeed the case, as monensin suppressed the appearance of fully glycosylated CPY (p2CPY) in CaCl 2 treated pmr1⌬ but not in WT cells (Fig. 6B, right).
Mn 2ϩ -sensitive mutants were found to be enriched in the functional category of vesicle-mediated transport including late endosome retrograde transport involving Tlg2 (48), a t-SNARE protein needed for the fusion of endosome-derived vesicles with the late Golgi (49,50). Based on this finding we wondered if the CaCl 2 -dependent suppression of CFW sensitivity and CPY glycosylation were impaired in pmr1⌬ tlg2⌬ double mutants. Indeed, although tlg2⌬ mutants did not display an obvious CPY glycosylation defect, CaCl 2 could not rescue CPY glycosylation defects and viability of CFW-treated pmr1⌬ tlg2⌬ double mutants (Fig. 6D). To further assess the role of CaCl 2 in vesicle transport, we analyzed different mutants defective in the coatomer or COPI coat required for the forma- tion of retrograde transport vesicles from the Golgi to the ER and between Golgi cisternae (intra-Golgi retrograde transport) (51,52). Again, CaCl 2 failed to rescue growth of pmr1⌬ mutants in the absence of Sec28 and in combination with mutations of Cop1 (ret1-1) or Cog3 (sec34 -2) (data not shown). Taken together, these results suggest that a functional intra-Golgi ret- APRIL 10, 2015 • VOLUME 290 • NUMBER 15 rograde transport is essential for the Ca 2ϩ -dependent bypass of pmr1⌬ glycosylation defects.

Intra-Golgi Manganese Redistribution
Finally, to more directly assess the idea that CaCl 2 stimulates intracellular vesicle trafficking, we used the exocytic SNARE Snc1 protein to monitor protein trafficking (53). The chimeric GFP-Snc1 protein is dynamically localized at the plasma membrane by continuous endocytic recycling, via endosomes, to the trans-Golgi, from where it is rapidly trafficked back to the plasma membrane. It has been previously shown that GFP-Snc1 accumulates in internal structures when Golgi function is blocked (54). Consistent with a known defect in Golgi function (31), the absence of Pmr1 leads to the redistribution of GFP-Snc1 to punctuated structures (Fig. 6E). The addition of CaCl 2 restored GFP-Snc1 localization to the cell surface, suggesting that CaCl 2 indeed rescues Golgi trafficking in pmr1⌬ mutant. By contrast, CaCl 2 addition could not restore the plasma membrane localization of GFP-Snc1 in a tlg2⌬ mutant background, which blocks the transport of GFP-Snc1 to the Golgi from endosomes. Therefore, these results strongly suggest that CaCl 2 promotes Golgi-vesicle trafficking overcoming the lack of Pmr1.

DISCUSSION
Here we dissect a remarkable mechanism by which CaCl 2 suppresses pleiotropic phenotypes linked to impaired cis-Golgi manganese transport ( Fig. 7; see the figure legend for an explanation). This mechanism relies on functional ER and late endosome/trans-Golgi Mn 2ϩ transport, and we provide evidence that calcium stimulates intra-organelle Mn 2ϩ redistribution through intracellular vesicle trafficking.
The P-type ATPase Spf1 and the Nramp transporter Smf2 are required for the CaCl 2 -mediated suppression of CFW sen-sitivity and CPY glycosylation. Spf1 and Smf2 activities might be required for ER and Golgi manganese supply and thus be required for vesicle-mediated manganese transport. Recently, the Spf1 has been shown to regulate Mn 2ϩ transport into the ER (14), and the addition of extracellular Ca 2ϩ accordingly suppressed SPF1 mutant phenotypes (35,55). Smf2 was predicted to transport Mn 2ϩ across membranes toward the cytosol by the assumption that Nramp transporters transport divalent cations in this direction (8). However, as is the case of Nramp1, the direction of the metal flux is still controversial (56). Thus, some authors propose that Nramp1 functions as a pH-dependent proton/divalent cation antiporter delivering divalent metal ions into acidic compartments (57)(58)(59). Accordingly, Nramp1, but not Nramp2, can rescue the metal ion stress phenotype of yeast mutants, suggesting that both proteins differ in the direction of transport (60). Notably, when expressed in yeast, Nramp1 localizes to the ER (data not shown) and thereby is unlikely to complement the transport activity of trans-Golgi-localized Smf2. Unfortunately, in contrast to other ions, studies on the abundance and intracellular distribution of manganese are hampered by the lack of chemical or genetically encoded manganese reporters (61).
In this work we specifically localize Smf2 in the late endosome/trans-Golgi, and based on our results, we believe that Smf2 might supply the trans-Golgi with Mn 2ϩ needed for the activity of mannosyltransferases such as Mnn1 (37,62). Neutralization of acidic trans-and post-Golgi compartments by monensin might alter Smf2 flux direction and, therefore, compromise CaCl 2 -dependent alleviation of CFW sensitivity. In addition, mutations in the vacuolar-type H ϩ -transporting FIGURE 6. Functional vesicle trafficking/fusion is essential for CaCl 2 -dependent rescue of pmr1⌬ glycosylation defects. A, illustration of the endomembrane system. Organelles (ER, Golgi, endosome, and vacuole) and secretory, endocytic, and CPY pathways are depicted. B, drop test sensitivity of WT, pmr1⌬, sec18 -20, and pmr1⌬ sec18 -20 against CFW (10 g/ml) without (top) or with (bottom) the addition of 10 mM CaCl 2 . Cells were grown in permissive (23°C, left) or semi-permissive (29°C, right) conditions. C, monensin, a drug that blocks intracellular transport, counteracts CaCl 2 -dependent suppression of glycosylation defects. Left, WT and pmr1⌬ cells were spotted onto YPAD, monensin (25 g/ml), CFW (10 g/ml), CFW ϩ CaCl 2 (10 mM), and CFW ϩ CaCl 2 ϩ monensin. Right, pulse-chase analysis of CPY maturation in WT and pmr1⌬ cells without or with the addition of CaCl 2 (10 mM) or CaCl 2 ϩ monensin (40 g/ml). ER (p1), Golgi (p2), and vacuole (m) CPY forms are indicated. D, pmr1⌬ mutants grown in the presence of CaCl 2 remain glycosylation deficient in the absence of Tlg2. Left, drop test sensitivity of WT, pmr1⌬, tlg2⌬ and pmr1⌬ tlg2⌬ cells against CFW (10 g/ml) without (top) or with (bottom) the addition of CaCl 2 (10 mM). Right, pulse-chase analysis of CPY maturation with or without the addition of CaCl 2 (10 mM). E, CaCl 2 rescues Snc1 protein trafficking. Plasma membrane localization of Snc1-GFP was quantified in WT, pmr1⌬, tlg2⌬ and pmr1⌬ tlg2⌬ cells grown without (white bars) or with the addition of CaCl 2 (10 mM, black bars). Bar, 5 m. Error bars represent S.D. Double asterisks (**) indicate p Ͻ 0.01. Phase contrast (Ph) and Snc1-GFP images are shown. ATPase (V-ATPase), which alter Golgi acidification, share multiple pmr1⌬ phenotypes (33). We find that Smf2 co-localizes with Atx2, a poorly characterized, putative trans-Golgi Mn 2ϩ transporter that could function in pumping Mn 2ϩ in the opposite direction to Smf2. Evidence for Atx2 ion transport activity is based on the observation that the protein shares functional characteristics with the SLC39 family of metal ion transporters (63). Consequently, Smf2 and Atx2 might form part of a late endosome/trans-Golgi Mn 2ϩ import/export system required for a stable equilibrium between Mn 2ϩ and other ions in the late endosome/trans-Golgi.
Regulation of Mn 2ϩ homeostasis is highly conserved between yeast and higher eukaryotes, and Mn 2ϩ transport enhancing mutations in the human ortholog of PMR1, ATP2C1 can protect mammalian cells from the cytotoxic effects of Mn 2ϩ (64). The contribution of defective Mn 2ϩ transport on Hailey-Hailey disease progression is still under debate. However, increasing evidence points to the possibility that impaired manganese homeostasis triggers keratinocyte differentiation (6) and causes genetic instability (2).
We first anticipated that CaCl 2 -dependent suppression of pmr1⌬ phenotypes could involve signal transduction pathways. However, this seem not to be the case, as CaCl 2 -mediated rescue of pmr1⌬ is not coupled to Ca 2ϩ /calmodulin-dependent changes in gene expression or protein re-localization. Increasing evidence links Ca 2ϩ to the regulation of membrane trafficking and fusion events (65,66). The precise mechanism by which calcium regulates membrane trafficking is still poorly understood. It has been proposed that transiently released luminal calcium is required to trigger the last stages of membrane fusion (23). Accordingly, the addition of CaCl 2 suppresses the vacuole fragmentation phenotype of pmr1⌬ mutants (33). In addition, calcium might also regulate the formation of intra-Golgi retrograde transport vesicles as it has been shown to stabilize COPI coat onto the Golgi membrane (67). The addition of CaCl 2 caused a significant increase in the intracellular calcium levels and might account for a permanent induction of retroand anterograde pathways. Along this line, we found that CaCl 2 decreased intracellular manganese levels and restored Golgito-cell surface recycling of the exocytic SNARE Snc1-GFP chimera in pmr1⌬ but not in pmr1⌬ tlg2⌬ mutants. These results point to the possibility that CaCl 2 promotes Golgi to trans-Golgi network, to secretory vesicle, to plasma membrane trafficking of vesicles. Based on our data we suggest that Mn 2ϩcontaining vesicles might emerge from the trans-or post-Golgi and fuse with the cis-Golgi, supplying the cis-Golgi with essential Mn 2ϩ for the action of sugar transferases. Consequently, Ca 2ϩ may also stimulate retro-and anterograde trafficking between later secretory pathway organelles and the ER. The results of this study raise the possibility that stimulation of vesicle transport in human cells can bypass ATP2C1 disease phenotypes or, yet more interestingly, can counteract neurotoxicity upon manganese exposure.