Localization-independent Regulation of Homocysteine Secretion by Phosphatidylethanolamine N -Methyltransferase*

Genetic ablation of phosphatidylethanolamine N -meth-yltransferase (PEMT) in mice causes a 50% reduction in plasma homocysteine (Hcy) levels. Because hyperhomocysteinemia is an independent risk factor for cardiovascular disease, resolution of the molecular basis for this reduction is of significant clinical interest. The PEMT pathway is a metabolically channeled process localized to the endoplasmic reticulum (ER). To assess the importance of PEMT localization for Hcy homeostasis, we identified and ablated the minimal ER targeting motif. Mutagenesis of a conserved, C-terminal lysine residue (197) relocalized the enzyme to the Golgi, demonstrating that Lys-197 is essential for targeting PEMT to the ER. To evaluate the functional significance of PEMT localization, hepatoma cell lines were generated that stably expressed either ER- or Golgi-localized PEMT only. Intriguingly, stable expression of PEMT in either the ER or the Golgi caused increased Hcy secretion. Moreover, PEMT-medi-ated Hcy secretion correlated with the methyltransferase activity of the enzyme, independently of subcellular localization. Thus, our data suggest that Hcy homeostasis is regulated concomitantly polyacrylamide standards (Bio-Rad). lowing polyvinylidene di-fluoride immunoblotted monoclonal anti-HA an- tibody (1:5000 dilution). peroxidase-conjugated secondary reagent (1:5000 dilution). to Biomax MR film (Kodak) at room

Elevated Hcy 1 levels are a risk factor for cardiovascular disease, in particular atherosclerosis and myocardial infarction (1,2). Additionally, meta-analyses of clinical studies have revealed that hyperhomocysteinemia is positively associated with an increased risk of cardiovascular disease and stroke independently of other risk factors (3)(4)(5)(6). Hyperhomocysteinemia is also an independent risk factor for Alzheimer disease and dementia (7). Although plasma Hcy levels are clearly of significant clinical interest, the molecular basis underlying Hcy homeostasis remains poorly understood.
Recently, we demonstrated that mice homozygous for a disrupted PEMT allele display a Ͼ50% reduction in circulating Hcy concentrations (8). Furthermore, primary hepatocytes cultured from PEMT null mice secrete ϳ50% less Hcy (8). Conversely, ectopic expression of PEMT in a cell line that lacks endogenous PEMT promotes Hcy secretion (8). In early studies, the synthesis of creatine by guanidinoacetate methyltransferase was proposed to be the major contributing reaction in homocysteine formation (9,10). Our data reveal a key, yet previously unrecognized, role for PEMT in Hcy homeostasis.
PEMT is a liver-specific enzyme that catalyzes three sequential transmethylation reactions in the production of phosphatidylcholine (PC) (11). S-Adenosylmethionine (AdoMet) is the methyl donor for each reaction and S-adenosylhomocysteine (AdoHcy) is the demethylated product, which is subsequently hydrolyzed to adenosine and the non-protein amino acid, Hcy (11,12). Hydrolysis of AdoHcy is carried out by the cytosolic enzyme, S-Adenosylhomocysteine hydrolase (AdoHcyase) (12,13). Efficient hydrolysis of AdoHcy is essential, as an increase in AdoHcy levels, along with the associated decrease in the cellular AdoMet/AdoHcy ratio, inhibits AdoMet-dependent methyltransferases (14).
Approximately 40 different mammalian methyltransferases, including DNA, RNA, protein, lipid, and small molecule methyltransferases, use AdoMet as a methyl donor and consequently produce AdoHcy during the methylation reaction (15,16). Because AdoHcy from each transmethylation reaction can potentially be used to produce Hcy, it is all the more striking that genetic ablation of just one methyltransferase, PEMT, suffices to depress circulating Hcy levels by half (8). The question thus arises: what molecular features of the PEMT pathway cause the preferential use of PEMT-derived AdoHcy as a substrate for AdoHcyase?
PEMT is localized in the ER and is present in a subfraction of ER membranes that co-fractionates biochemically with mitochondria, known as mitochondria-associated membranes (17,18). The mitochondria-associated membranes represent a metabolically important region of the ER, characterized by a significant enrichment in lipid biosynthetic enzymes relative to other areas of the ER (19 -21). Because the PEMT pathway is also a metabolically channeled process (22), we investigated whether precise subcellular localization might be essential to the role of PEMT in Hcy homeostasis.
To evaluate the importance of PEMT localization for Hcy homeostasis, we relocalized PEMT to a different cellular sub-compartment by defining and ablating the ER retrieval motif. The canonical retrieval motif for ER membrane proteins features a lysine residue at the Ϫ4, Ϫ3 (KKXX) or Ϫ5, Ϫ3 (KXKXX) positions relative to the C-terminal end (23). This motif functions in mammals, yeast, and plants (24 -26) through interactions with specific subunits of the Cop I complex (27). Here, we have validated the minimal ER targeting motif of PEMT and have presented biochemical evidence that PEMT can promote Hcy secretion through an ER localization-independent mechanism.

Materials
Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum, restriction endonucleases, and Platinum TM Pfx DNA polymerase were from Invitrogen. Oligonucleotides were synthesized at the Institute of Biomolecular Design, University of Alberta. S-adenosyl-L-[methyl-3 H]methionine (15 Ci/mmol) was obtained from Amersham Biosciences. Non-radiolabeled S-adenosyl-L-methionine and anti-HA monoclonal antibody were from Sigma. FuGENE 6 TM transfection reagent was from Roche Applied Science.
Phosphatidylmonomethylethanolamine was from Avanti Polar Lipids. Anti-protein disulfide isomerase (PDI) antibody was from Stressgen Biotech. Mannosidase II (Man II) and TGN-38 antibodies were kindly provided by Dr. Paul Melançon, University of Alberta. Goat anti-rabbit and goat anti-mouse secondary antibodies were from Pierce. Texas Red-X goat anti-mouse IgG, Alexa Fluor 488 goat anti-rabbit, and Prolong Antifade TM reagent were from Molecular Probes.

Recombinant Plasmid Construction
Plasmids encoding PEMT localization mutants were generated by PCR-based mutagenesis using the hemagglutinin (HA)-hPEMT-pCI plasmid as template (18). This plasmid encodes a previously described HAtagged derivative of the human PEMT protein, which is enzymatically active (18). To mutate the residues of the putative ER retrieval motif, a common HA-PEMT-specific sense primer and a different antisense primer for each mutant were used. Primer sequences are available upon request. Mutant PCR products were subcloned into pCI (Promega) using XhoI and XbaI restriction sites. Transcription from the pCI vector is under the control of a CMV promoter. All constructs were sequenced at the Molecular Biology Services Unit, University of Alberta.

Cell Culture, Transient Transfections, and Generation of Stable Cell Lines
Cell lines were obtained from the American Type Culture Collection repository. NRK (normal rat kidney) cells were maintained in DMEM, 10% fetal bovine serum, 100 units/ml penicillin, and 100 g/ml streptomycin sulfate at 37°C, 5% CO 2 . McArdle RH-7777 hepatoma cells were similarly maintained, except the culture media also included 10% horse serum. Transient transfection of NRK cells was performed using the FuGENE 6 TM reagent according to the manufacturer's instructions. McArdle RH-7777 stable transfectants were generated using the FuGENE 6 TM reagent or by the calcium phosphate precipitation method (28).

Confocal Microscopy Analysis
NRK cells were seeded onto 22-mm coverslips in 6-well plates at a density of 1 ϫ 10 5 cells/well. Following overnight incubation at 37°C, 5% CO 2 , the cells were transfected with plasmids encoding mutant PEMT derivatives as described above. 24 h post-transfection, cells were serum-starved for 1 h and fixed in 4% paraformaldehyde/phosphatebuffered saline, pH 7, for 10 min. Following permeabilization with 0.2% Triton X-100/phosphate-buffered saline for 2 min, cells were incubated with the first primary polyclonal antibody at 37°C for 1 h at the indicated dilution: PDI (1:100), Man II (1:300), or TGN-38 (1:200). Cells were then incubated with the second primary antibody (monoclonal anti-HA, 1:150) for 1 h at 37°C. Cells were subsequently incubated for 1 h at 37°C with both secondary antibodies: Texas Red-X goat antimouse (1:200) and Alexa Fluor 488 goat anti-rabbit (1:200). Coverslips were mounted onto slides with Prolong Antifade TM reagent and immunofluorescence was visualized using a Zeiss LSM510 confocal microscope. All images presented are single sections in the Z plane.

PEMT Enzymatic Assays
In Vitro PEMT Assays-These assays were performed as described previously (29). Briefly, cells were washed and harvested into phosphate-buffered saline, pelleted at 1000 ϫ g, and resuspended in buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA). Following homogenization by sonication, protein homogenates (50 g) were assayed for PEMT activity using phosphatidylmonomethylethanolamine as a methyl acceptor and S-adenosyl-L-[methyl-3 H]methionine as the methyl group donor.

Immunoblot Analysis
Proteins from cell homogenate (5 g) were separated by Tris-glycine SDS-polyacrylamide gel electrophoresis on 12.5% polyacrylamide gels calibrated with prestained molecular weight standards (Bio-Rad). Following electrophoresis, proteins were transferred to polyvinylidene difluoride membranes and immunoblotted with monoclonal anti-HA antibody (1:5000 dilution). Protein-antibody complexes were detected by enhanced chemiluminescence with horseradish peroxidase-conjugated secondary antibody using the ECL reagent (Amersham Pharmacia Biosciences) as directed (1:5000 dilution). Membranes were exposed to Biomax MR film (Kodak) at room temperature.

Statistical Analysis
Statistical differences between the means of two groups were determined using a Student's t test. Mean values of more than two groups were compared using a one-way analysis of variance followed by a Student-Newman-Keuls post-hoc test.

Identification of an ER Retrieval
Motif-To identify a putative ER retrieval motif, comparative analysis of known PEMT primary sequences was performed. A classical C-terminal ER retrieval motif (KKXX) is present in yeast and neurospora PEMT orthologs (Fig. 1A). Although the functional relevance of this motif is unknown, only the lysine residue at the Ϫ3 position (Lys-197 in humans) is conserved in higher species (Fig.  1A). Either lysine in a di-lysine motif can be mutated to an arginine or histidine residue without detriment to ER targeting (32). Because Lys-197 is flanked by histidine and arginine residues (HKRX), one of several combinations of basic residues could target PEMT to the ER (Fig. 1A). To serve as a functional ER retrieval signal, the C-terminal motif should be exposed to the cytosol (24,25). PEMT adopts a topography within the ER membrane that positions both termini in the cytosol (Fig. 1B) (18). Thus, Lys-197 of human PEMT is optimally positioned to interact with the ER retrieval machinery.
Mutation of Lys-197 Prevents Targeting of PEMT to the ER-To investigate the role of the putative retrieval motif in targeting PEMT to the ER, we generated recombinant constructs in which Lys-197 and the flanking basic residues, His-196 and Arg-198, were mutated to serine residues. Each of the constructs included a N-terminal HA tag, because the epitope recognized by the PEMT antibody spans the region that includes the putative retrieval motif (17) and mutations in this region abolish epitope recognition. 2 HA-tagged PEMT derivatives have been characterized, and the tagged enzyme retains significant transmethylation activity compared with the unmodified enzyme (18).
To assess the role of specific residues in targeting PEMT to the ER, constructs encoding the mutant PEMT proteins were transfected into NRK cells, and colocalization studies were performed using the ER marker, PDI. NRK cells, which do not express endogenous PEMT protein, 2 are a commonly used model for subcellular localization studies (33,34). As expected, the unmodified PEMT enzyme co-localized with the ER protein, PDI ( Fig. 2A). In contrast, a PEMT derivative in which both Lys-197 and Arg-198 were mutated to serine residues (K197S/ R198S) failed to colocalize with PDI; instead, the double mutant displayed a juxtanuclear localization (Fig. 2B). Mutation of His-196 resulted in a PEMT derivative that was either poorly expressed or rapidly degraded, as H196 transfectants were very rarely detected. 2 Thus, the role of His-196 in targeting PEMT to the ER could not be determined.
To determine whether Lys-197 and Arg-198 are both required for retrieval of PEMT to the ER, each residue was individually mutated to serine residues, generating the constructs K197S and R198S. Confocal analysis demonstrated that the R198S mutant co-localized with PDI, suggesting that Arg-198 is not essential for ER localization (Fig. 2C). In contrast, mutation of Lys-197 caused a loss of colocalization with PDI (Fig. 2D). Moreover, the K197S (Fig. 2B) mutant adopted a juxtanuclear localization similar to that of the K197S/R198S double mutant (Fig. 2B). Therefore, mutation of Lys-197 is sufficient to abrogate ER targeting of PEMT, supporting the notion that Lys-197 is essential for retrieval of PEMT to the ER.
PEMT-K197S Mutants Are Localized to the Golgi-PEMT derivatives that harbor a mutation of Lys-197 (K197S/R198S or K197S) adopt a juxtanuclear localization that resembles the pattern of Golgi. To define the localization of the displaced K197S mutants, we performed co-localization analysis with the Golgi enzyme Man II. Extensive co-localization between the Lys-197 mutants and Man II was observed, suggesting that mutation of Lys-197 relocalizes PEMT to the Golgi (Fig. 3, B  and D). Conversely, the unmodified enzyme and the R198S mutant, each of which colocalized with PDI, did not colocalize with Man II (Fig. 3, A and C). Thus, Lys-197 is essential for targeting PEMT to the ER, as mutagenesis causes relocalization to the Golgi.
Golgi-localized PEMT Catalyzes PC Biosynthesis-Identification of Lys-197 as a key residue in targeting PEMT to the ER enabled us to engineer PEMT variants that are localized to distinct regions of the cell. By modulating the subcellular localization of the enzyme, the significance of PEMT localization in Hcy homeostasis could thus be investigated. To directly assess the functional significance of PEMT localization, McArdle RH-7777 cell lines were generated that stably express either ER-or Golgi-localized PEMT. Mutation of Lys-197 also caused the relocalization of PEMT from the ER to the Golgi in McArdle RH-7777 cells. 2 McArdle hepatoma cells are a frequently used model for the study of lipid metabolism and energy homeostasis and do not express endogenous PEMT (8,35,36).
PEMT catalyzes the AdoMet-dependent methylation of PE to produce PC (11). To determine whether the Golgi-localized enzymes are active, PEMT enzymatic assays were performed. Cells expressing PEMT localization mutants were radiolabeled with metabolic precursors, and the radiolabeling of PE and PC was quantitated.
As expected, stable expression of the unmodified enzyme caused a significant increase in the PC/PE ratio (Fig. 4A). Similarly, expression of the ER-localized R198S mutant resulted in a marked increase in the PC/PE ratio, demonstrating that mutation of Arg-198 is not inhibitory for PEMT activity (Fig. 4A). Surprisingly, stable expression of each of the Golgilocalized PEMT mutants (K197S/R198S and K197S) also caused a significant increase in the PC/PE ratio compared with cells stably transfected with empty vector (Fig. 4A). Thus, displacement of PEMT from the ER does not abolish the methyltransferase activity in cells. Furthermore, localization of PEMT in the Golgi does not preclude the methylation-based synthesis of PC. Although the ER-and Golgi-localized PEMT derivatives displayed similar activity levels in cells (Fig. 4A), immunoblot analysis revealed that amounts of the Golgi-localized enzymes were in fact significantly lower than the ERlocalized PEMT derivatives (Fig. 4B). These data suggest that Golgi-localized PEMT displays higher specific activity than the corresponding ER-localized enzyme. Alternatively, PC biosyn-thesis may be limited either through product-based feedback inhibition or via an unknown PC-sensing regulatory system.
PEMT Promotes Hcy Secretion Independently of Subcellular Localization-PEMT catalyzes three sequential methylation reactions in the biosynthesis of PC (Fig. 5A) (11). Generation of a PC molecule results in the concomitant production of three molecules of AdoHcy, each of which can be hydrolyzed to yield Hcy (Fig. 5A) (12). In the next series of experiments we investigated whether PEMT promotes Hcy secretion through a localizationdependent mechanism. Media were collected from cells that stably expressed ER-or Golgi-localized PEMT, and Hcy levels were measured. As predicted, stable expression of the ER-localized enzymes (PEMT and R198S) resulted in a significant increase in media Hcy content (Fig. 5B). However, stable expression of the Golgi-localized PEMT mutants (K197S/R198S and K197S), also significantly increased the secretion of Hcy compared with cells that were stably transfected with empty vector (Fig. 5B). Thus, these data suggest that PEMT promotes Hcy secretion through a localization-independent mechanism.
Hcy Secretion Increases Concomitantly with PEMT Activity-To further define the molecular determinants of PEMT-dependent Hcy homeostasis, we investigated whether Hcy secretion is modulated coordinately with PEMT activity. Cell lines that stably expressed various levels of ER-localized PEMT or the Golgi-localized derivative, K197S, were identified by in vitro PEMT assays (Fig. 6A). To test the hypothesis that Hcy secretion is regulated coordinately with PEMT activity, media Hcy content from the different cell lines was measured.
As before, expression of either the ER-or Golgi-localized PEMT enzymes increased the media Hcy content (Fig. 6B). Because a PEMT-dependent increase in Hcy secretion was evident in all stable cell lines (each represents a distinct clone), it is unlikely that the promotion of Hcy secretion by ER-or Golgilocalized PEMT is due to genomic integration effects. Notably, Hcy secretion was augmented with increasing PEMT activity. Moreover, Hcy secretion was strongly correlated with methyl-

FIG. 5. Expression of PEMT in the ER or Golgi promotes Hcy secretion.
A, interrelationship between PEMT-catalyzed biosynthesis of PC and Hcy metabolism. PEMT catalyzes the synthesis of PC via three AdoMet-dependent transmethylation reactions. AdoHcy produced with each methylation step is hydrolyzed by AdoHcyase to Hcy and adenosine (Ado) (11,12). Hcy undergoes further metabolic processing to generate cysteine or AdoMet or is exported from the cell (42). B, total Hcy content in cellular media was assayed using a high-performance liquid chromatography-based method 18 h after plating McArdle RH-7777 hepatoma cells that stably express wild-type PEMT or PEMT localization mutants. Results are expressed as the mean of three separate experiments (each performed in duplicate) Ϯ S.E. transferase activity of both the ER (R 2 ϭ 0.7637)-and Golgilocalized (R 2 ϭ 0.9364) enzymes (Fig. 6C). Therefore, PEMT promotes Hcy secretion through a localization-independent mechanism, and Hcy secretion correlates with PEMT activity.

DISCUSSION
Plasma Hcy concentrations elicit significant clinical interest because elevated levels are associated with coronary heart disease, stroke, peripheral arterial occlusive disease, Alzheimer disease, and dementia (3,(5)(6)(7). A number of animal models have been engineered that display mild or severe hyperhomocysteinemia; for example, mice that are heterozygous or homozygous for a disruption of the methylenetetrahydrofolate reductase (MTHFR) allele display a 1.6-and 10-fold elevation in plasma Hcy levels, respectively (37). Notably, both heterozygous and homozygous MTHFR-deficient animals display abnormal lipid deposition in the proximal portion of the aorta (37). Mice that are heterozygous or homozygous for a disruption of cystathionine ␤-synthase (CBS) allele display a 2-fold and 40-fold elevation in plasma Hcy levels respectively (38). Homozygous null CBS mice present with hepatic microvesicu-lar steatosis and die within 5 weeks of birth, whereas heterozygous CBS animals exhibit impairment of endothelium-dependent vasodilation (38,39).
In contrast, we recently demonstrated that genetic ablation of PEMT results in a Ͼ50% reduction in plasma Hcy levels (8). This phenotype highlighted a previously unrecognized role for PEMT in Hcy homeostasis. However, the underlying nature of PEMT-dependent Hcy homeostasis remains unclear. Because a mechanistic understanding of PEMT in Hcy homeostasis may aid in the development of novel therapies for hyperhomocysteinemia, the objective of our current study was to elucidate the molecular basis of PEMT-dependent Hcy secretion.
The liver is the primary site of PEMT expression, with extrahepatic PEMT accounting for a mere fraction of corporeal expression (11,17,40,41). Traditionally, the liver and kidney were considered the major regulatory tissues for plasma Hcy homeostasis (42). However, although the significance of the liver in Hcy homeostasis was recently reaffirmed (43), data from the CBS transgenic mouse support a lesser role for the kidney than previously proposed (44). Thus, the liver-specific expression profile of PEMT may be optimal for a role in Hcy homeostasis.
PEMT is localized in the ER of the liver and in a lipid metabolism-rich region of the ER known as mitochondria-associated membranes (17,18). Given the subcellular location of the enzyme and the fact that the PEMT pathway is a metabolically channeled process (22), we investigated whether PEMT localization is essential for Hcy homeostasis. However, several factors supported the notion that ER localization might be superfluous to the role of PEMT in Hcy homeostasis. PEMT is an integral membrane protein that uses PE and AdoMet as substrates (11,45); PE is present in all subcellular membranes and AdoMet is distributed throughout the cytosol (45). Furthermore, AdoHcyase, which hydrolyzes PEMT-derived AdoHcy to supplement the circulating Hcy pool, is also present throughout the cytosol (46). Because the key elements in the PEMT-dependent production of Hcy are not spatially delimited, we rationalized that PEMT might modulate Hcy homeostasis irrespective of subcellular localization.
In this study, we have demonstrated that the conserved C-terminal Lys-197 residue (Ϫ3 position) is essential for targeting PEMT to the ER, as mutagenesis caused the mislocalization of the enzyme to the Golgi. Approximately 23% of ER membrane proteins (46 of 202) are predicted to be targeted to the ER by a C-terminal di-lysine motif, but only 3.5% of ER membrane proteins (7/202) feature a single lysine residue at the Ϫ3 position (47). The role of this lysine residue in targeting proteins other than PEMT to the ER remains to be determined.
As predicted, Golgi-localized PEMT retains methyltransferase activity, supporting our hypothesis that a correctly folded enzyme catalyzes the reactions of the PEMT pathway irrespective of localization. Furthermore, expression of PEMT in either the ER or the Golgi promoted Hcy secretion. This result is significant insofar as it suggests that it is PEMT, and not a PEMT-dependent ancillary factor in the ER, that causes the altered plasma Hcy levels in PEMT knock-out mice; mutation of PEMT Lys-197 would not be expected to relocalize such an ancillary factor to the Golgi.
Large quantities of PC are produced in the liver to replenish the hepatic pools used for production of bile and very low density lipoproteins (48). PEMT accounts for 30% of hepatic PC biosynthesis (22,49,50), which translates to significant levels of Hcy production, because 3 molecules of Hcy are generated with each molecule of PC synthesized. We cannot rule out the possibility that a perturbation of hepatic membrane lipid composition alters the secretion of Hcy or the reuptake of an intermediary metabolite such as methionine, in PEMT knock-out and K197S (D-F)) was measured after 18 h of incubation. C, correlation between Hcy secretion and PEMT-specific activity. Filled diamonds represent data from three separate experiments with three different cell lines that stably express wild-type PEMT. Filled triangles represent data from three separate experiments with three different cell lines that stably express PEMT-K197S. mice. Nevertheless, it is likely that plasma Hcy levels are decreased because of the drastic reduction in levels of PEMTderived Hcy. This notion is further supported by our finding that Hcy secretion is strongly correlated with PEMT activity and hence with PC biosynthesis.
Previously, we identified key motifs in PEMT that bind AdoMet/AdoHcy and resolved the topography of the enzyme (51). Knowledge of the membrane topography of PEMT enabled us to map the AdoMet/AdoHcy binding sites to the cytosolic portion of the enzyme. Golgi-localized PEMT displays methyltransferase activity, indicating that the mislocalized enzyme likely adopts the correct topography within the membrane. Such a topographical organization may prove central to the role of PEMT in Hcy homeostasis, as it facilitates access to both the cytosolic pool of AdoMet and the soluble enzyme AdoHcyase (45,46). Thus, a key determinant of PEMT-mediated Hcy secretion may be the spatial organization of PEMT within the ER membrane rather than the specific subcellular localization of PEMT. Furthermore, although the reactions of the PEMT pathway are metabolically channeled, orientation of the PEMT active site in the cytosol with AdoHcyase may represent an additional level of efficiency in Hcy production. Intriguingly, recent findings suggest that the yeast genes encoding phospholipid biosynthesis enzymes and AdoHcyase are coordinately regulated at the transcriptional level (52). Thus, coordinate regulation of PEMT and AdoHcyase, whether at the transcriptional level or through spatial juxtaposition of enzymatic activities, may be an evolutionary conserved facet of Hcy homeostasis.
In summary, we have demonstrated that Lys-197 is essential for targeting PEMT to the ER, as mutation of this conserved residue relocalized the methyltransferase to the Golgi. Golgilocalized PEMT not only catalyzes the reactions of the PEMT pathway but also promotes secretion of Hcy. Moreover, Hcy secretion correlates with the activity of both ER-and Golgilocalized PEMT. Because PEMT modulates plasma Hcy homeostasis, the enzyme represents a novel target for therapeutic intervention in patients with hyperhomocysteinemia. Resolution of the molecular basis of PEMT-dependent Hcy homeostasis may aid in the development of such novel approaches and therapeutic agents.