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PROTEIN AND VESICLE TRAFFICKING, CYTOSKELETON

Cytoplasmic targeting signals mediate delivery of phospholemman to the plasma membrane

Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri

Submitted 9 March 2005 ; accepted in final form 13 December 2005

	ABSTRACT

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The FXYD protein family consists of several small, single-span membrane proteins that exhibit a high degree of homology. The best-known members of the family include the -subunit of the Na⁺-K⁺-ATPase and phospholemman (PLM), a phosphoprotein of cardiac sarcolemma. Other members of the family include corticosteroid hormone-induced factor (CHIF), mammary tumor protein of 8 kDa (Mat-8), and related to ion channels (RIC). The exact physiological roles of the FXYD proteins remain unknown. To better characterize the function of the members of the FXYD protein family, we expressed several members of the family in Madin-Darby canine kidney (MDCK) cells. All of the FXYD proteins, with the exception of PLM, were primarily found in the basolateral plasma membrane. Surprisingly, PLM, a previously characterized plasma membrane protein, was found to colocalize with the endoplasmic reticulum marker protein disulfide isomerase. Treatment of MDCK cells expressing PLM with an agonist of PKC caused some of the PLM to be redistributed to the plasma membrane. Site-directed mutagenesis of residues within the cytoplasmic domain of PLM indicated that a negative charge at Ser69 is necessary to shift the localization of PLM to the plasma membrane. In addition, other regions of PLM necessary for either its endoplasmic reticulum or plasma membrane localization have been elucidated. In contrast to PLM, the plasma membrane localization of CHIF and RIC was not altered by mutation of potential cytoplasmic phosphorylation sites. Overall, these results suggest that phosphorylation of specific residues of PLM may direct PLM from an intracellular compartment to the plasma membrane.

protein trafficking; FXYD proteins

View larger version (74K): [in this window] [in a new window]   Fig. 1. Alignment of FXYD family members. Alignment of the deduced amino acid sequences of the human and rat -subunits of the Na+-K+-ATPase, mouse phospholemman (PLM), rat corticosteroid hormone-induced factor (CHIF), mouse mammary tumor protein of 8 kDa (Mat-8), and protein related to ion channels (RIC). Invariant amino acids are shown shaded. A bar indicates the putative transmembrane domain. The boxed glutamate residue in PLM marks the amino terminus of the mature protein after cleavage of the signal sequence.

Several members of the FXYD family appear to associate with the Na⁺-K⁺-ATPase to modulate its activity (reviewed in Ref. 14). The Na⁺-K⁺-ATPase is a membrane-associated protein responsible for the high intracellular K⁺ and low intracellular Na⁺ concentrations characteristic of most animal cells. The enzyme consists of two noncovalently linked subunits: a 100-kDa multispanning membrane protein termed the -subunit, and the -subunit, a smaller glycosylated membrane protein. Multiple isoforms for both - (₁, ₂, ₃, ₄) and - (₁, ₂, ₃) subunits have been identified. These isoforms exhibit a tissue-specific and developmental pattern of expression that may be important in the maintenance and regulation of Na⁺-K⁺-ATPase activity (reviewed in Ref. 8). Although not integral parts of the Na⁺-K⁺-ATPase enzymatic complex, several FXYD proteins modulate Na⁺-K⁺-ATPase activity. For example, the -subunit can modify the voltage dependence of K⁺ activation (5) and influence the apparent affinity of the enzyme for Na⁺, K⁺, and ATP (2, 50). When expressed in Xenopus oocytes, PLM induces a slight decrease in the K⁺ affinity and a nearly twofold decrease in the Na⁺ affinity of the ₁₁ and ₂₁ isozymes (15). In addition, in native rat heart, it appears that PLM can associate with the ₁ but not the ₂ subunit (19). Moreover, PLM-deficient mice have reduced cardiac Na⁺-K⁺-ATPase activity (28). In contrast, CHIF when expressed in Xenopus oocytes with the Na⁺-K⁺-ATPase ₁- and ₁-subunits, increases the Na⁺ affinity and decreases the apparent K⁺ affinity of the enzyme (3). Finally, FXYD7 can interact with the -isozymes containing the ₁ but not the ₂ isoform. Moreover, in oocytes, FXYD7 decreases the apparent K⁺ affinity of the ₁₁ and ₂₁ isozymes, but not ₃₁. However, in the rat brain, FXYD7 appears to associate only with the ₁ isozymes (4).

To better characterize the function of the members of the FXYD protein family, we have expressed several of the family members in a mammalian cell line. To determine the subcellular localization of the different family members, we fused a myc epitope tag to the carboxy terminus of each of the FXYD proteins and expressed them in Madin-Darby canine kidney (MDCK) cells. All of the FXYD proteins, with the exception of PLM, were found primarily in the basolateral plasma membrane. Surprisingly, PLM, a previously characterized plasma membrane protein, was found to colocalize with the endoplasmic reticulum (ER) marker protein disulfide isomerase (PDI) in the MDCK cells. Treating MDCK cells expressing PLM with an agonist of PKC caused some of the PLM to be redistributed to the plasma membrane. Site-directed mutagenesis of residues within the cytoplasmic domain of PLM indicated that a negative charge at Ser69 is necessary to shift the localization of PLM to the plasma membrane. In addition, other regions of PLM necessary for either its ER or plasma membrane localization have been elucidated. These results suggest that phosphorylation of specific residues of PLM may direct PLM from an intracellular compartment to the plasma membrane. Understanding the regulation of PLM may provide insights into the physiological roles of PLM at the plasma membrane.

	MATERIALS AND METHODS

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Antibodies. The ₁-subunit of the Na⁺-K⁺-ATPase was identified with an antibody to a synthetic peptide derived from the amino-terminus of the rat ₁-subunit (7). PDI was identified with a commercially available rabbit polyclonal antibody (StressGen Biotechnologies, Victoria, BC, Canada). T7 peptide antibody was purchased from Biodesign International (Saco, ME). The mouse monoclonal antibody raised against the myc epitope (17) was kindly provided by Maurine E. Linder (Washington University).

Cell culture. MDCK cells (clone G) were obtained from W. J. Nelson (Stanford University). The cells were maintained by being passaged at low density every 3 days. Following trypsinization, the cells were resuspended in Dulbecco's modified Eagle's medium supplemented with 10% FBS, 200 µM L-glutamine, 0.25 µg/ml Fungizone, 100 U/ml penicillin, and 100 µg/ml streptomycin. The cells were transfected with the indicated cDNAs subcloned into pcDNA3.1 (–) Myc-HisA (Invitrogen, Carlsbad, CA) such that all expressed polypeptides included a myc epitope tag at the carboxy terminus. PCR was used to create all constructs from the following cDNAs: PLM (accession no. NM019503), CHIF (L41254 [GenBank] ), Mat-8 (BF300582 [GenBank] ), RIC (U72680 [GenBank] ), and a (NM_145717 [GenBank] ). All cDNAs expressed in MDCK cells were verified by sequencing. For some constructs, a T7 peptide epitope (MASMTGGQQMG) tag replaced the myc tag at the carboxy terminus. This was to verify that the charges associated with the myc epitope tag did not affect cellular localization. In all cases, proteins with either the myc or T7 peptide tag yielded identical results. MDCK cells were transfected using Lipofectamine (GIBCO-BRL, Rockville, MD) according to the supplier's protocol. Stably transfected cells were selected for and grown in media supplemented with 800 µg/ml geneticin (GIBCO-BRL). Briefly, following transfection, the cells were split and plated at a high density. The cells were grown for 1 wk or more without being passaged; the medium was replaced approximately every 2 days. For PKA stimulation, confluent MDCK cells on coverslips were treated with 2 mM dibutyryl adenosine 3',5'-cyclic monophosphate (DbcAMP; Sigma, St. Louis, MO) in growth medium. At the indicated times, the treated cells were washed once in 150 mM NaCl and 25 mM HEPES, pH 7.4 (HBS), and processed for immunofluorescence. To activate PKC, cells were treated with 1 µM phorbol 12-myristate 13-acetate (PMA; Sigma) in growth medium. At the times indicated, the cells were washed once in HBS and processed for immunofluorescence.

Immunofluorescence. MDCK cells were plated at low density on collagen-coated coverslips and grown to confluence. Once confluent, the MDCK cells were washed once with HBS and fixed/permeabilized in 100% methanol at –20°C for 10 min. The coverslips were washed three times with 4°C HBS. Nonspecific sites were blocked for 1 h at 37°C in HBS containing 5% normal goat serum (NGS). Primary antibodies against the -subunit of the Na⁺-K⁺-ATPase (₁ diluted 1:100) and myc (ascites fluid diluted 1:2,000) were diluted in HBS containing 1% NGS and applied to cells overnight at 4°C. Following three washes in HBS, Alexa 488-, or Cy3-conjugated secondary antibodies (Molecular Probes, Eugene, OR) diluted 1:1,000 in HBS containing 0.1% NGS, were applied for 1 h at room temperature. The samples were washed three times in HBS and mounted in ProLong Antifade (Molecular Probes). The samples were viewed with a Zeiss Axioskop microscope (x40 objective). Images were captured digitally with the microscope using Axiovision 2.0 software. Images were viewed and assembled using PhotoShop 6.0 and Illustrator 9.0 (Adobe).

	RESULTS

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PLM is localized to ER in MDCK cells. To study the expression of an FXYD protein in a polarized, mammalian cell background, we expressed PLM in MDCK epithelial cells, a frequently used renal cell culture model. To identify the PLM polypeptide, a myc epitope tag was fused to the cytoplasmic carboxy terminus of the protein sequence. As shown in Fig. 2, indirect immunofluorescence with an antibody to the myc epitope indicated that PLM is restricted to an intracellular compartment in the MDCK cells. Surprisingly, PLM colocalized with an ER chaperone protein, PDI, and exhibited little overlap with the Na⁺-K⁺-ATPase -subunit at the plasma membrane. No fluorescence was observed in MDCK cells transfected with only the myc vector (data not shown). As shown, PLM with a T7 epitope tag also was restricted to intracellular compartments demonstrating that the myc sequence did not affect PLM localization. These results are in contrast to earlier studies, in which PLM has been characterized as the major plasma membrane substrate for PKA and PKC in heart cells (41). In addition, following activation of PKA or PKC, PLM has been identified in the plasma membrane of cardiac (6, 41, 43), skeletal (55, 56), smooth muscle (10), and liver (13) cells, as well as in adrenal tumor cells (57).

View larger version (85K): [in this window] [in a new window]   Fig. 2. In Madin-Darby canine kidney (MDCK) cells, PLM colocalizes with the luminal endoplasmic reticulum (ER) chaperone protein, protein disulfide isomerase (PDI). Transfected MDCK cells expressing myc- (top and middle) or T7 (bottom)-tagged PLM were stained with an anti-myc or anti-T7 antibody and visualized using a Cy3-conjugated goat anti-mouse antibody. Top, to identify the plasma membrane, cells were costained with an antibody to the -subunit of the Na+-K+-ATPase. The -subunit antibody was visualized with an Alexa 488-conjugated goat anti-rabbit antibody. Middle, the cells were costained with an antibody to the ER protein PDI. Endogenous PDI expression was visualized with an Alexa 488-conjugated goat anti-rabbit antibody. To demonstrate overlap of PLM and PDI or the -subunit, the respective images were merged; yellow indicates regions of colocalization. Bar, 5 µm.

View larger version (109K): [in this window] [in a new window]   Fig. 3. PLM is the only FXYD family member located in the ER of transfected MDCK cells. Transfected MDCK cells expressing the indicated myc-tagged FXYD family member were processed for double-immunofluorescence microscopy using a monoclonal antibody to myc and a rabbit antibody to the -subunit of the Na+-K+-ATPase. The myc antibody was detected using a Cy3-conjugated goat anti-mouse antibody and the -subunit antibody was visualized with an Alexa 488-conjugated goat anti-rabbit antibody. To demonstrate overlap, the respective images were merged; yellow indicates regions of colocalization. Bar, 10 µm.

The exact functions and cellular location of Mat-8 or RIC have not been determined. Interestingly, both Mat-8 and RIC were originally identified as factors upregulated by oncogenes. Both proteins localized to the basolateral plasma membrane of MDCK cells (Fig. 3). Mat-8 RNA is expressed in primary human breast tumors and in breast tumor cell lines (36), and murine Mat-8 is expressed at high levels in the uterus, stomach, and colon. RIC gene expression has been detected in the heart, spleen, lung, testis, and skeletal muscle of normal adult rats (18). It is not clear whether these upregulated genes are required for the transformed state or whether these proteins are characteristic features of secretory epithelial cells and are simply maintained in the transformed state.

The carboxy terminus of PLM is sufficient for ER localization. The localization of PLM to the ER appears to be unique among the FXYD family. Because the primary amino acid structure of the FXYD proteins is conserved (Fig. 1), chimeras were used to determine the domain of PLM responsible for its intracellular localization. Moreover, because the cytoplasmic domain of PLM is more conserved among species than its amino-terminal, extracellular domain, we replaced or added portions of the PLM cytoplasmic domain to another FXYD family member, (Fig. 4A). As shown in Fig. 4B, when the cytoplasmic domain of was replaced with the cytoplasmic domain of PLM (_1–47/PLM_38–72), the chimera localized to the same intracellular compartment as wild-type PLM. To further delineate the region responsible for the intracellular localization of PLM, the last 15 amino acids of the PLM carboxy terminus were added to the full-length -protein (/PLM_56–72). As shown in Fig. 4B, localization was shifted to the ER. Both constructions resided in the same intracellular compartment as the wild-type PLM protein implying that the carboxy terminus of PLM is sufficient to confer localization to the ER.

View larger version (49K): [in this window] [in a new window]   Fig. 4. Chimeras of a and the PLM carboxy terminus are localized in an intracellular compartment. A: extracellular and membrane domains of (1–47) were fused to the cytoplasmic domain of PLM (38–72) or the full-length sequence was fused to the extended carboxy-terminal portion of PLM (56–72). B: immunofluorescence of MDCK cells expressing a, PLM, or chimeras of a and PLM. Expressed proteins were detected using an anti-myc antibody and visualized using a Cy3-conjugated goat anti-mouse antibody. Bar, 10 µm.

View larger version (72K): [in this window] [in a new window]   Fig. 5. Expression of PLM truncation mutants in MDCK cells reveals domains important for ER retention. Immunofluorescence of MDCK cells expressing truncations of the wild-type PLM protein tagged with a myc epitope at the carboxy terminus. PLM was truncated after Glu57 (PLM1–57) or after Gly51 (PLM1–51). The truncated PLM proteins fused to a myc epitope were detected with a monoclonal myc antibody and visualized using a Cy3-conjugated goat anti-mouse antibody. The plasma membrane was detected using a polyclonal antibody to the -subunit of the Na+-K+-ATPase. The -subunit antibody was visualized with an Alexa 488-conjugated goat anti-rabbit antibody. To demonstrate overlap the respective images were merged; yellow indicates regions of colocalization. While PLM1–51 is retained in the ER, PLM1–57 colocalizes with the -subunit of the Na+-K+-ATPase at the plasma membrane. Bar, 10 µm.

View larger version (103K): [in this window] [in a new window]   Fig. 6. Activation of PKC changes the subcellular localization of PLM to the plasma membrane. MDCK cells expressing PLM as a myc-tagged fusion protein were removed from culture before treatment (time 0) or after the addition of 1 mM PMA at a specified time (5, 20, 40, or 60 min). After treatment, the cells were fixed and processed for immunofluorescence. PLM was detected using a monoclonal antibody to myc and visualized using a Cy3-conjugated goat anti-mouse antibody. The plasma membrane was detected using a polyclonal antibody to the -subunit of the Na+-K+-ATPase. The -subunit antibody was visualized with an Alexa 488-conjugated goat anti-rabbit antibody. To demonstrate colocalization, the respective images were merged; yellow indicates regions of overlap. After 20 min of treatment, a significant amount of PLM was detected at the plasma membrane. Bar, 5 µm.

View larger version (78K): [in this window] [in a new window]   Fig. 7. Activation of PKC changes the subcellular localization of the protein/PLM chimera (/PLM56–72) to the plasma membrane. MDCK cells expressing /PLM56–72 as a myc-tagged fusion protein were removed from culture before treatment (A) or 40 min after the addition of 1 mM PMA (B). After treatment, the cells were fixed and processed for immunofluorescence. /PLM56–72 was detected using a monoclonal antibody to myc and visualized with the use of a Cy3-conjugated goat anti-mouse antibody. After 40 min of treatment, a significant amount of the /PLM chimeric protein was detected at the plasma membrane.

Phosphorylation of PLM is required for plasma membrane localization. To determine which residues are important for the change in PLM localization, we substituted negatively charged aspartates where PLM could be phosphorylated by PKC. Mutation of a phosphorylated amino acid to aspartic or glutamic acid often mimics the phosphorylation event (21). As shown in Fig. 8, when Ser62, Ser63, Ser68, and Ser69 were replaced with aspartates (PLM_DDDD), the modified PLM was redirected to the plasma membrane. The ability of the PLM_DDDD mutant to transport to the plasma membrane supports phosphorylation as an important mechanism for the proper trafficking of PLM.

View larger version (100K): [in this window] [in a new window]   Fig. 8. MDCK cells expressing PLM with mutations at Ser62, Ser63, Ser68, and/or Ser69. A: MDCK cells expressing the indicated myc-tagged PLM mutants were detected using a monoclonal antibody to myc and visualized with the use of a Cy3-conjugated goat anti-mouse antibody. Wild-type PLM, PLMGAAA, PLMADDA, PLMDDDA, PLMGADD, and PLMGAAD were localized to intracellular compartments, whereas the PLMDDDD and PLMGDDD mutants were delivered to the plasma membrane. B: MDCK cells expressing the T7-tagged PLM mutants were detected using an antibody to the T7 epitope and visualized using a Cy3-conjugated goat anti-rabbit antibody. Bar, 10 µm.

View larger version (46K): [in this window] [in a new window]   Fig. 9. MDCK cells expressing mutants of PLM truncated at residue 69. MDCK cells expressing the indicated PLM mutants were detected using a monoclonal antibody to myc and visualized using a Cy3-conjugated goat anti-mouse antibody. Wild-type PLM1–69 and PLMGAAD1–69 were localized at the plasma membrane, whereas the PLMGAAA1–69 mutant remained in an intracellular compartment. Bar, 10 µm.

View larger version (62K): [in this window] [in a new window]   Fig. 10. Charge on cytoplasmic Ser170 does not influence the localization of RIC. Immunofluorescence was performed on MDCK cells expressing mouse RIC tagged with a carboxy-terminal myc epitope. RIC was detected using a monoclonal antibody to myc and visualized using a Cy3-conjugated goat anti-mouse antibody. The plasma membrane was detected using a polyclonal antibody to the -subunit of the Na+-K+-ATPase and visualized with an Alexa 488-conjugated goat anti-rabbit antibody. RIC is detected at the plasma membrane, regardless of mutations to the only residue capable of being phosphorylated. Bar, 5 µm.

	DISCUSSION

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The cytoplasmic domain of PLM from the canine, rat, human, and mouse is almost completely conserved, with the exception that mouse PLM has a serine at position 69 instead of a threonine. However, this is a conserved change because this threonine fits the phosphorylation consensus sequence for PKC (25). Although it is curious that threonine phosphorylation has not been observed in vitro from peptides derived from canine PLM (54), recent results using antibodies raised against PLM phosphopeptides suggest that T69 is phosphorylated (24).

In Xenopus oocytes, coexpression of PKA and PLM increased the amplitude of the ion current observed and the amount of PLM present at the plasma membrane (38). When PKA was coexpressed with a phosphorylation-defective PLM mutant (SSST 62, 63, 68, 69 AAAA), there was no increase in the amplitude of the induced ion current or the amount of PLM at the membrane. Therefore, phosphorylation by PKA appears to have an effect on the membrane localization of PLM. Even in the absence of PKA, the same study showed a PLM mutant (S68A) in the oocyte membrane in greater amounts than wild-type PLM. Coexpression of PKA and the same mutant (S68A) further increased the amount of PLM in the oocyte membrane. In contrast, PLM SSST 62, 63, 68, and 69 AAAA was not visible in plasma membranes, and coexpression with PKA did not increase the amplitude of ion current induced or the amount at the plasma membrane. These results suggest that Ser68 plays a role in directing PLM to the oocyte membrane; however, other serine residues also influence PLM localization.

Phosphorylation as a mechanism for release from ER. Phosphorylation and dephosphorylation of proteins is an important mechanism for regulating cellular processes. In addition, phosphorylation events may regulate the cellular trafficking of proteins. In addition to PLM, the involvement of phosphorylation for trafficking from the ER has been proposed for another protein with a highly charged cytoplasmic tail, the Iip35 isoform of the major histocompatibility complex class II-associated invariant chain (Ii). In B-cell lines, the ER retention of Iip35 is ineffective and a fraction of Iip35 is transported through the Golgi complex associated with class II molecules in a phosphorylation-dependent manner (27). Mutations altering the charge of Ser6 or Ser8 in the cytoplasmic tail of Iip35 (M¹HRRRSRSCREDQKPV) to an alanine prevented detection of the mutants outside the ER. Presumably, the change in protein charge caused by phosphorylation effectively negates the ability of the RRR motif to retain Iip35 in the ER.

Several signals that influence the trafficking of membrane proteins through the ER have been identified. For example, a stretch of negatively charged amino acids appears to act as a signal for release from the ER (39), whereas the RXR motif acts as an ER retention signal (30). However, in both of these cases, the proteins, VSVG and K⁺ channels, respectively, required the formation of homotrimers or subunit assembly for proper cellular function. Without knowing the functional activity of PLM, it is difficult to determine whether oligomerization is required for proper function. In addition to these signals, a carboxy-terminal valine residue appears to function as an ER export signal in several proteins, including FXYD7 (16, 22, 52). However, FXYD7 is the only FXYD family member with a carboxy-terminal valine, so this export signal does not play a role in trafficking of the other FXYD proteins. The simplest explanation for our findings suggests that PLM is sequestered in the ER by the carboxy-terminal RRR motif until cellular stimulation activates second messengers, leading to the phosphorylation of PLM. Presumably, phosphorylated PLM is subsequently recruited to the plasma membrane, where it can associate with other proteins to perform its physiological function.

PLM may be phosphorylated by kinases other than PKC or PKA. PLM derived peptides are an excellent substrate for NIMA kinase (29), the product of a cell cycle-regulatory gene, or myotonic dystrophy protein kinase (37). Moreover, coexpression of myotonic dystrophy protein kinase and PLM in Xenopus oocytes reduces the amount of PLM at the plasma membrane. The relevance of these interactions in vivo has not been determined; however, these observations do suggest that multiple kinases may act on PLM. Lu et al. (29) proposed that PKA phosphorylation of PLM inhibits further phosphorylation by PKC. However, we were unable to detect any difference in the final localization of PLM at the plasma membrane when the cells were stimulated with PKA activators before PKC activators, or vice versa (data not shown). Previous results suggest that Ser68 is phosphorylated by PKA stimulation (54), and this residue has been implicated in mediating oocyte membrane localization (38). When we attempted to express a PLM_AADA mutant in MDCK cells, protein expression could not be detected despite cell growth in the selection antibiotic (data not shown). Phosphorylation at multiple sites on PLM may affect the kinetics of the subcellular shift to the plasma membrane or the ability of PLM to associate with other factors.

Potential physiological implications of multiple PLM phosphorylation events. Previous results have identified phosphorylated PLM in purified canine cardiac sarcolemmal vesicles after stimulation of the -adrenergic receptor with isoproterenol (42). Our findings are consistent with these observations. Furthermore, the variety of combinations with which PLM may be phosphorylated suggests a role for PLM in the diverse responses observed between tissue types after adrenaline stimulation. Our study is the first to demonstrate that phosphorylation of Ser69 is important for PLM localization to the plasma membrane. Moreover, the association of PLM to other proteins at the plasma membrane or ER may be influenced by its pattern of phosphorylation. There is evidence to suggest that the pattern of PLM phosphorylation is both important and variable for its cellular function. In isolated skeletal muscle, insulin regulates the phosphorylation of both Ser63 and Ser68, whereas adrenaline regulates phosphorylation of Ser68 (54).

The increased transport of phosphorylated PLM to the plasma membrane may reflect its ability to override retention signals to exit the ER. Alternatively, phosphorylation of PLM may allow it to interact with another factor and traffic to the plasma membrane. This model is consistent with the identification of a 14-3-3 protein associating with Iip35 in a phosphorylation-dependent manner to mediate its exit from the ER (27). Understanding of the regulation and role of PLM phosphorylation may provide insights into the functions of PLM and its putative protein partners.

	GRANTS

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This work was supported by National Institutes of Health Grants GM-39746 and DK-064704.

	FOOTNOTES

 

Address for reprint requests and other correspondence: R. W. Mercer, Dept. of Cell Biology and Physiology, Washington Univ. School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110 (e-mail: rmercer{at}cellbio.wustl.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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