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Originally published In Press as doi:10.1074/jbc.M309760200 on October 21, 2003

J. Biol. Chem., Vol. 279, Issue 4, 3003-3013, January 23, 2004
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Epitope Tagging of the Yeast K+ Carrier Trk2p Demonstrates Folding That Is Consistent with a Channel-like Structure*

Ge-Fei Zeng{ddagger}, Marc Pypaert§, and Clifford L. Slayman{ddagger}

From the {ddagger}Department of Cellular and Molecular Physiology and the §Center for Cell and Molecular Imaging, Yale University School of Medicine, New Haven, Connecticut 06520

Received for publication, September 3, 2003 , and in revised form, October 20, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
TRK family proteins, which mediate the concentrative uptake of potassium by plant cells, fungi, and bacteria, resemble primitive potassium channels in sequence and have recently been proposed actually to fold like potassium channels in a 4-MPM motif (Durell, S. R., and Guy, H. R. (1999) Biophys. J. 77, 789 - 807), instead of like conventional substrate porters in the 12-TM motif (Gaber, R. F., Styles, C. A., and Fink, G. R. (1988) Mol. Cell. Biol. 8, 2848-2859). The known fungal members of this family possess a very long hydrophilic loop, positioned intracellularly in the K+-channel model and extracellularly in the substrate porter model. This and two shorter hydrophilic segments have been tested as topological markers for the true folding pattern of TRK proteins using Saccharomyces cerevisiae Trk2p. Hemagglutinin epitope tags were inserted into all three segments, and the enhanced green fluorescent protein (EGFP) was fused to the C terminus of Trk2p. The gene constructs were expressed from a high copy plasmid, and sidedness of the tags was determined by native fluorescence (EGFP), indirect immunofluorescence, and immunoelectron microscopy. Both the long-loop tag and the C-terminal EGFP fusion allowed abundant protein to reach the plasma membrane and support normal yeast growth. In all determinations, the long-loop tag was localized to the inner surface of the yeast cell plasma membrane, thus strongly supporting the channel-like folding model. Additional observations showed (i) membrane-associated Trk2p to lie in proteolipid rafts; (ii) significant tagged protein, expressed from the plasmid, to be sequestered in cytoplasmic vesicular-tubular clusters; and (iii) suppression of such clusters by yeast growth in 5-10% glycerol. This chaperone-like effect may assist other membrane proteins (overexpressed or heterologously expressed) to function within the yeast plasma membrane.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The primary mechanism for potassium accumulation in most animal cells is countertransport for sodium mediated by the "sodium pump" or Na,K-ATPase. In non-animal cells, including plants, fungi, and bacteria, which lack the sodium pump as an obligatory system, at least three other mechanism have been demonstrated to operate under various conditions. These include 1) ATP-fueled potassium uptake mediated by bacterial "Kdp" ATPases, which are related to (but distinct from) the Na,K-ATPase (1, 2); 2) channel-mediated potassium accumulation (3-5) driven by the large resting membrane voltages that are common among plants and free-living microorganisms (3, 6); and 3) carrier-mediated cotransport of potassium with protons (7-9) or sodium (10-13). These cotransporters draw energy not only from the large membrane voltage in these organisms, but also from chemical gradients of the cotransported ions; and they can concentrate potassium from rarefied environments (such as pond water or certain laboratory broths) to ratios of 10,000 or more (7).

The latter mechanism now appears to be the most prevalent and rests upon a family of proteins that are widely distributed among plants, fungi, and bacteria, but seem to be absent from animal systems. These are the so-called TRK proteins, for which the first gene to be sequenced was, indeed, TRK1 from yeast (Saccharomyces cerevisiae) (14). There are now approximately four dozen known members of this family, and they have attracted special attention because of sequence similarities to bacterial channels for potassium ions (15-18).

The yeast S. cerevisiae actually possesses two closely related variants of the TRK protein, designated Trk1p and Trk2p, which were initially described as 12-TM proteins, containing 12 membrane-spanning {alpha}-helices, on the basis of hydropathy analysis (14, 19). This picture was consistent with the general notion that these presumed coupled-ion transport proteins would structurally resemble the major families of amino acid, sugar, and toxic agent transporters, which also catalyze ion-coupled transport. More recently, however, sequence similarities between the TRK family and bacterial potassium channels, along with the emergent crystal structure for KcsA, the K+-channel from Streptomyces lividans (20), have prompted construction of a 4-MPM model for the TRK proteins (21) in which the core of the protein would be an internal tetramer of paired membrane-spanning helices bounding four re-entry loops, which penetrate the membrane from the outside surface. Bar-and-string diagrams of these two models (with several important loci indicated; see below) are compared in Fig. 1 for S. cerevisiae Trk2p, the smaller of the two yeast proteins.



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  		FIG. 1.
Bar-and-string diagrams contrasting two possible folding maps for yeast Trk2p. Upper, 12-TM map with 12 putative transmembrane helices based on a simple hydropathy plot of the primary sequence as first described by Ko and Gaber (19). The long hydrophilic loop (37% of the total protein) would lie at the extracellular surface of the plasma membrane. Lower, 4-MPM map showing four pairs of transmembrane helices, each pair sandwiching a re-entry loop (P-loop), which would enter and exit the membrane through the extracellular surface. Each pair of transmembrane helices with its P-loop would be analogous to the core (MPM) motif of bacterial potassium channels. In this map, the long hydrophilic loop would lie intracellularly. Insert sites tested for the hemagglutinin epitope (red triangles) and for the fusion with EGFP (green ellipses; C-terminal) are indicated, showing the expected sidedness shifts of HA insertions between the two maps. Certain potential regulatory sites bearing histidines (pink squares), protein kinase A (PKA) motifs (dark-blue circles), and protein kinase C (PKC) motifs (light-blue circles) are also shown. Sc., S. cerevisiae.

 
Although the functional literature on potassium transport in Saccharomyces is complex and rather skeltered, the major observations have been that (a) under different physiological conditions, potassium can be accumulated via the TRK proteins either with high affinity (K0.5 ~ 30 µM) or with moderate affinity (K0.5 ~ 10 mM) and also via a non-TRK pathway with low affinity (K0.5 ~ 60 mM) (22); and (b) based on single gene disruptions, Trk1p (but not Trk2p) is essential for high affinity uptake. These facts have naturally led to the hypothesis that Trk1p should be a high affinity K+ transporter and Trk2p a moderate affinity transporter.

The additional fact that wild-type Saccharomyces can concentrate potassium by >10,000-fold from dilute media indicates either that the yeast resting membrane voltage is well negative to -200 mV or that the high affinity transporter must couple potassium influx with the flux of another ion species. Potassium transport in yeast has long been known to show two-site or multisite kinetics (23, 24), and the most important "second-site" ion, at least in acidic media, has been assumed to be protons: one entering with each K+ ion. This notion was propelled by general similarities between high affinity K+ transport in Saccharomyces and that in Neurospora, for which proton-potassium cotransport is well documented (7, 8, 25). However, TRK proteins in both bacteria and plants have been shown to transport sodium along with potassium (12, 13), and a more satisfactory current view of the Saccharomyces TRK proteins is that one or both are two-site transporters in which the "first" site has a narrow specificity for K+, whereas the "second" site can bind almost any available monovalent cation, including H+, K+, Na+, and Cs+ (24, 26-28).

The molecular mechanism for coupling ion movements to the uptake of neutral substrates has been under intense study for several proteins, especially the lactose transporter of Escherichia coli, which couples proton influx with influx of a variety of {beta}-galactoside molecules and which is the best understood 12-TM transporter (29). Even for this protein, it has not been possible to separate the intuitive notion that species as different as H+ and lactose should pass through different "channels" within the protein from the structural picture of a single hydrophilic cavity, now demonstrated by crystallography (30). The possibility that the TRK proteins might be folded like a simple potassium channel but at the same time might couple the movement of K+ with such different ions as H+ or Na+ raises a number of important questions. Among them are where parallel channels might reside in such a molecule and whether and how a single channel undergoes cyclic changes in affinity as alternative ions transit (21).

It is therefore important to establish clearly which of the two existing models (12-TM or 4-MPM) is realistic for the TRK proteins. We have approached this problem by attaching epitope labels at several points in S. cerevisiae Trk2p and then examining the whole cell distribution of the protein by immunofluorescence and immunoelectron microscopy. The results clearly support the channel-like 4-MPM model over the earlier 12-TM model.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Strains
S. cerevisiae haploid strain HY483 (kindly provided by Dr. R. F. Gaber, Northwestern University) was used throughout this study for expression of plasmid-borne variants of the TRK2 gene. Both the TRK1 and TRK2 genes are deleted in this strain. Haploid strain YPH499 and plasmid pYGW1 (purchased from Research Genetics, Huntsville, AL) were used for cloning the TRK2 gene, and E. coli strain DH10B was used for plasmid propagation. The strain genotypes are as follows: HY483, MAT{alpha} ade2-1 can1-100 GAL+ his3-11,15 leu2-3,112 SUC2 trk1{Delta}::HIS3 trk2k{Delta}::HIS3 trp1-1 ura3-1; YPH499, MAT{alpha} his3-{Delta}200 lys2-801amber leu2-{Delta}1 ade2-101ochre trp1-{Delta}63 ura3-52; DH10B, F' mcrA {Delta}(mrr-hsd RMS-mrcBC) {phi}80dlacZ {Delta}M15 {Delta}lacX74 deoR recA1 endA1 araD139 {Delta}(ara,leu)7697 galU galK {Lambda} rpsL nupG; and pYGW1, <- -2µ-Ori-Gal1P{blacktriangleright} --[-EcoRI-BxtXI-HindIII--]-ColE1-{blacktriangleleft}Amp-{blacktriangleleft}URA3-->, where the polylinker, [...],is shown with the three restriction sites (of 13) that were used, and the end arrows indicate completion of the circle.

Growth Media, Buffers, and Reagents
Five different growth media were used. For routine preparation of cells, YPD (1% yeast extract and 2% Bacto-peptone broth with 2% dextrose) as described by Sherman (31) was used. For recovery of recombinant clones, SCM (synthetic complete medium, with glucose as carbon source) was used (31). For the immunofluorescence experiments, with robust production of Trk2p, SCM lacking uracil, for plasmid retention, but containing either 2% raffinose or 2% galactose (SCRaf and SCGal, respectively) as carbon source was used and supplemented with 100 mM KCl. Finally, for growth tests of cells carrying tagged Trk2p, LSRaf and LSGal (the low-salt medium devised by Gaber et al. (14), again using either 2% raffinose or 2% galactose as carbon source) was used.

The buffers used for handling DNA (i.e. for PCR, restriction cutting, and ligation) were those recommended by the manufacturer for each reagent. All buffers used for immunofluorescence and immunoelectron microscopy began with phosphate-buffered saline (PBS1; 50 mM K+ phosphate (pH 7.5) and 150 mM NaCl), to which fixatives, detergents, stabilizers, and antibodies were added.

DNA Enzymes—The restriction endonucleases BstXI, NcoI, HindIII, and EcoRI, along with T4-DNA ligase, were obtained from New England Biolabs (Beverly, MA); and pfx DNA polymerase was from Invitrogen.

Primary Antibodies—Monoclonal mouse anti-hemagglutinin (HA) IgG (16B12) was purchased from Berkeley Antibody Co. (Richmond, CA) and rabbit anti-green fluorescent protein (GFP) IgG from Molecular Probes (Eugene, OR).

Secondary Antibodies—Fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG, TRITC-conjugated goat anti-mouse IgG, and TRITC-conjugated goat anti-rabbit IgG were obtained from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). "Cappel" rabbit anti-mouse IgG came from ICN Pharmaceutical (Aurora, OH), and Staphylococcus protein A complexed with 5-nm gold ("5-nm protein A-gold") was bought from the Department of Cell Biology, Utrecht University (The Netherlands).

Detergents—Triton X-100 and Tween 20 came from American Bioanalytical (Natick, MA).

Electron Microscopy-grade Reagents—Formaldehyde, paraformaldehyde, glutaraldehyde, methylcellulose, and uranyl acetate were purchased from Electron Microscopy Sciences (Ft. Washington, PA).

Ordinary Chemicals—Most chemicals were reagent-grade and bought from either Sigma or Calbiochem. Citifluor mounting medium was obtained from TedPella, Inc. (Redding, CA).

Growth Measurements
Growth measurements for potassium sufficiency testing were carried out via a modified standard drop test as described by Bihler et al. (22). Cells were pre-grown to OD600 = 1 in 30 °C shaking cultures in LSRaf medium; and the resultant log-phase cells were harvested by centrifugation, rinsed twice with double-distilled water, and resuspended in water at OD600 = 1. Serial 10-fold dilutions of this suspension were made and frogged or dropped (7 µl) onto 2% agar plates of K+-supplemented LSGal medium. The plates were incubated for 2 days at 30 °C and then recorded on a flat-bed digital scanner.

Cloning of TRK2
The GeneWeaverTM I kit (Research Genetics) was used to clone TRK2. Yeast genomic DNA was isolated from strain YPH499, and 10 ng was used to amplify TRK2 by PCR with primers 1 and 2 (see Table I), in which the first segments are plasmid-targeting sequences and the second represent the 5'- and 3'-ends of TRK2. The PCR product was cotransformed along with the BstXI-linearized pYGW1 vector into YPH499 by the lithium acetate method recommended in the GeneWeaver protocol. Recombinant clones were selected on synthetic complete medium dropout plates (without uracil), and plasmid DNA was isolated from yeast and transformed into E. coli strain DH10B for amplification. After re-isolation of plasmid DNA, the presence of the TRK2 gene—and its correct sequence—were confirmed by DNA sequencing. The recombinant plasmid pYGW1-TRK2 was then transformed into HY483 for expression of TRK2 under the control of the GAL1 promoter.


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  		TABLE I
PCR primers for cloning of yeast Trk2p and for epitope and fusion protein tagging

 
Insertion of the Influenza HA Epitope
Three sites were chosen for insertion of the HA epitope (YPYDVP-DYA): site I, between Ala264 and Asn265 (790GCA/AAC795); site II, between Thr487 and Arg488 (1459ACG/AGG1464); and site III, between Thr800 and Asn801 (2398ACC/AAC2403). These sites were selected as random-coil segments of the protein, computed via the PROTEAN algorithm in the LaserGeneTM software package (DNASTAR, Inc., Madison, WI), which should switch localization between the inside surface of the membrane and the outside (sites II and III) from the 12-TM model to the 4-MPM model or vice versa (site I), as shown in Fig. 1. The PCR primers used to construct the insertions are detailed in Table I.

For the site I insertion (Ala264-HA-Asn265), primers 3 and 4 plus pYGW1-TRK2 were used to make the initial segment (bp 1-792) of TRK2 with the HA tag overhung 3'; likewise, primers 5 and 6 were used to make the second segment (bp 793-1452) of TRK2 with HA overhung 5'. These two segments were then denatured by boiling and annealed to produce a single segment of TRK2 (bp 1-1452) with the HA tag inserted between bp 792 and 793. This was amplified by PCR with primers 3 and 6, and the product was digested with EcoRI and NcoI to yield the 5'-half of TRK2 (bp 1-1447) with the HA tag inserted between bp 792 and 793 and with 17 bp of 5'-plasmid sequence. The remainder of TRK2 (bp 1448-2670) was produced from pYGW1-TRK2 by NcoI/HindIII digestion, and these two were ligated with EcoRI/HindIII-digested pYGW1. The complete ligation product was transformed into E. coli DH10B, and the amplified recombinant plasmid was isolated and sequenced.

The site III insertion (Thr800-HA-Asn801) was accomplished in a completely analogous manner using primers 9-12. The site II insertion (Thr487-HA-Arg488) was begun by PCR of pYGW1-TRK2 with primers 7 and 8. The product was then cloned into the pCR-BluntII plasmid, excised with NcoI/HindIII, and ligated with the EcoRI/NcoI segment of TRK2 and EcoRI/HindIII-cut pYGW1.

Construction of the TRK2-EGFP Fusion Gene
On the basis of general experience, the C terminus of Trk2p was chosen as the most likely location for a non-disruptive fusion with the EGFP tag. PCR primers for the purpose are listed at the bottom of Table I. The EGFP gene was amplified by PCR from plasmid pEGFP-C1 (Clontech) with primers 13 and 14, leaving 21 bases homologous to the 3'-prime end of TRK2 (without the stop codon) overhung 5'. The 3'-half of TRK2 (bp 1447-2667) was amplified by PCR with primers 15 and 16, leaving 21 bases homologous to the 5'-end of the EGFP overhung 3'. These PCR products were mixed, denatured by boiling, and annealed; and the resultant segment was amplified via primers 14 and 15 to give the recombinant TRK2 half-gene fused to the EGFP gene. The fusion product was digested with NcoI and HindIII and then ligated with the EcoRI/NcoI segment of TRK2 and EcoRI/HindIII-cut pYGW1, as described above for the HA insertions. The ligation product was transformed into E. coli DH10B for amplification, and the recombinant plasmid pYGW1-TRK2-EGFP was isolated and sequenced.

Construction of TRK2-HA-EGFP
This was managed completely analogously to the construction of pYGW1-TRK2-EGFP, except that the TRK2-HA (site I) segment excised from pYGW1 (EcoRI/NcoI; see above) was ligated, instead of the wild-type EcoRI/NcoI segment.

Immunofluorescence
Growth—Cells were grown overnight in SCRaf to OD600 ~ 1, harvested by centrifugation, rinsed with distilled water, and transferred to SCGal medium at 30 °C for a 4-h induction.

Fixation—The induced culture was injected with formaldehyde to a final concentration of 2% and then incubated at 25 °C with gentle shaking for 10 min. Cells were harvested by centrifugation, resuspended in PBS containing 3.7% formaldehyde, shaken for 90 min at 25 °C, rinsed twice with PBS, and resuspended in PBS plus 1.2 M sorbitol.

Spheroplasting—After another centrifugation, the cells were resuspended in protoplast buffer (PBS, 1.2 M sorbitol, 25 mM {beta}-mercaptoethanol, and 350 mg/ml of zymolyase 20T) at 5 x 108 cells/ml and incubated at 37 °C for 30 min. The resultant cells/spheroplasts were spun down, washed once with sorbitol buffer, and then resuspended in fresh PBS.

Permeabilzation—0.1% Triton X-100 was added to the fixed cell suspension in PBS, and the mixture was placed on ice for 10 min.

At this stage, 20 µl of suspended protoplasts (fixed-only or fixed permeabilized) was dropped into each well of a multiwell slide (Carlson Scientific, Peotone, IL), which had been precleaned with ethanol and coated with 0.1% poly-L-lysine (>70,000 kDa). 10 min was allowed for the spheroplasts to settle and adhere before further processing.

All antibody and staining reactions were carried out either in PBS plus 1 mg/ml bovine serum albumin for fixed-only cells or in the same solution plus 0.1% Tween 20 for fixed permeabilized cells following the general procedures described by Garnier et al. (32). The primary antibodies (mouse anti-HA IgG and rabbit anti-GFP IgG) were diluted 1:1000 in PBS and incubated with the protoplasts for 1 h at room temperature (~23 °C). The wells were washed with the suspension buffer (three times for 5 min each) to remove unreacted primary antibody and then reacted for 1 h with the appropriate secondary antibody (added at 1:200 dilution). Finally, the wells were washed again, filled with Citifluor (glycerol and antifade compound), and sealed under a coverslip. Slides were examined and photographed with a Zeiss Axiovert fluorescence microscope and/or a Zeiss LSM510 confocal microscope.

Immunoelectron Microscopy
Fixation and Sectioning—Cells (fixed and spheroplasted as described above) were refixed for 48 h at 4 °C in 8% paraformaldehyde and PBS, spun down, rinsed twice with PBS, and embedded in 10% bovine gelatin and PBS. Slices of the pellet (0.5 mm) were infiltrated overnight at 4 °C in sucrose (2.3 M in PBS) and then frozen in liquid nitrogen. Frozen sections (600 Å) were cut on a Leica Ultracut ultramicrotome and collected on Formvar/carbon-coated grids.

Immunolabeling—For HA detection, the grids were incubated with mouse anti-HA IgG (as described above) diluted 1:50 in PBS containing 1% fish skin gelatin, washed with PBS, and incubated with rabbit anti-mouse IgG at a dilution of 1:50. For EGFP detection, the grids were incubated with rabbit anti-GFP IgG at a dilution of 1:40 in PBS with 1% fish skin gelatin and then washed with PBS. Finally, both samples were incubated with 5-nm protein A-gold at a dilution of 1:70. After several washes with PBS, the sections were fixed for 5 min in 1% glutaraldehyde and PBS, washed with distilled water, and incubated in 1.8% methylcellulose and 0.5% uranyl acetate for 10 min on ice. Thereafter, the grids were air-dried and examined with a Tecnai-12 electron microscope.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Surface Localization of Trk2p via the C-terminal EGFP Tag—Because Trk2p directly supports potassium uptake from extracellular medium into the cytoplasm and contains many suitably hydrophobic stretches, it can reasonably be supposed to be resident in the yeast plasma membrane. This supposition was easily confirmed by the cellular distribution of EGFP fluorescence produced by expressing the plasmid (pYGW1)-borne fusion construct TRK2-HA-EGFP in the trk1{Delta} trk2{Delta} yeast strain HY483. As illustrated in Fig. 2 (upper panel), apart from occasional brightly stained cytoplasmic clumps, the majority of EGFP fluorescence was localized at cell surfaces in all experiments; and, as is illustrated in four of the stained cells of Fig. 2, this surface-localized fluorescence tended also to be punctate. The nature of both the cytoplasmic clumps and the apparent membranous clusters will be discussed in connection with immunoelectron microscopy (see Figs. 6, 7, 8).



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  		FIG. 2.
EGFP-tagged Trk2p is located at the yeast surface membrane. Shown is a comparison of a native fluorescence image for the fusion protein made by TRK2-HA-EGFP (upper panel) and the corresponding differential interference contrast (DIC) image of the same cells (lower panel). Note the bright surface ring on most cells; the distinct but low level fluorescence of some central vacuoles; and the occasional very intense staining of small cytoplasmic particles, which represent disorganized membranes resembling VTCs (see Fig. 8). Sequential images were made on a Zeiss Axiophot microscope equipped with fluorescence and differential interference contrast optics.

 



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  		FIG. 6.
Protein A-gold, bound either to anti-HA antibody (A and B) or to anti-GFP antibody (C), locates predominantly at the membrane inner surface. 600-Å frozen sections of yeast suspensions were post-reacted with antibodies and protein A-gold, refixed briefly with glutaraldehyde, stained with uranyl acetate, air-dried, and examined under an electron microscope (see "Experimental Procedures"). A and B are independent sections from separate experiments with anti-HA antibody, and C is from another experiment with anti-GFP antibody.

 



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  		FIG. 7.
Tagged Trk2p is not extractable from yeast total membranes by Triton X-100, satisfying the standard criterion for proteins bound to lipid rafts. 60 OD units of yeast cells (~6 x 108 cells) was disrupted with glass beads in sorbitol buffer and protease inhibitors and spun at low speed to remove unbroken cells and large debris. A sample (~100 ng of protein) from the low speed supernatant was retained for SDS-PAGE (Total, left lane), and the remainder was mixed and incubated for 30 min with iced 2% Triton X-100. After a high speed spin (100,000 x g for 1 h), another supernatant sample (also ~100 ng of protein) was removed for PAGE (SN, middle lane). The high speed pellet was then dissolved in 1% SDS, and a 50-ng sample was similarly processed for PAGE (Pellet, right lane). The positions of molecular mass standards (in kilodaltons) are shown to the left.

 



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  		FIG. 8.
Electron microscopic details of Trk2p expression from the pYGW1 plasmid marked by protein A-gold-labeled anti-HA antibody. A, two linked VTCs showing skeltered tubules and vesicles, often surrounded by multiple gold particles. These structures are the presumed repositories for excess Trk2p protein and clearly represent the bright cytoplasmic spots observed by fluorescence microscopy (Figs. 2, 3, and 5). B, a likely transition figure between VTCs and cortical ER. C, cortical ER laden with gold-marked protein and stacked loosely inside the plasma membrane. Such arrangements could easily be confused, upon fluorescence microscopy, with bona fide membrane rafts.

 
Because the C terminus of Trk2p was predicted to lie intracellularly in both folding models, this location for the EGFP fusion protein served as a controlled topological test, which could be analyzed by comparing the native fluorescence of EGFP with that produced by indirect staining with fluorescent antibody. Careful fixation with aldehyde mixtures does not generally degrade cell plasma membranes sufficiently to admit antibody-sized proteins (33, 34). The experiment of Fig. 3 (compare lower middle and upper middle panels) demonstrates that only detergent-permeabilized cells fluoresced with anti-GFP antibody, so the EGFP tag was indeed located at the inner surface of the plasma membrane. The fluorescence images for native GFP and anti-GFP antibody in detergent-permeabilized cells proved nearly identical everywhere, except for occasional intense staining of small bud cells (Fig. 3, compare lower middle and lower left panels). As expected, anti-GFP antibody showed negligible reaction with yeast proteins in cells transfected with the empty vector (Fig. 3, right panels) whether or not the membranes were treated with detergent.



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  		FIG. 3.
Anti-GFP antibody decorates the Trk2p-HA-EGFP fusion protein only in detergent-permeabilized cells. Left panels, native fluorescence of EGFP showing predominant distribution at the cell surfaces before detergent treatment (upper panel) and after detergent treatment (lower panel); middle panels, fluorescence of TRITC-conjugated anti-GFP antibody, which also showed predominant distribution at cell surfaces, but only after cells had been permeabilized by 0.1% Triton X-100; right panels, control cells expressing the empty plasmid pYGW1 and demonstrating complete lack of anti-GFP antibody reaction against native yeast proteins.

 
Topological Placement of the "Working" HA Tag, Inserted into the Long Hydrophilic Loop—Two of the three sites tested for insertion of the HA epitope, the "short segment" insertions at Thr487-Arg488 and Thr800-Asn801 (Fig. 1), caused Trk2p to be dispersed in clumps throughout the cytoplasm (data not shown), but it did not reach the plasma membrane sufficiently to generate satisfactory fluorescence images. Growth tests in 3 and 10 mM potassium demonstrated that, at high expression levels, some of the tagged Trk2p did become physiologically functional; but no way was found to simplify the fluorescence image for these two inserts, and the constructs were abandoned. Thus, topological orientation could not be determined for the peptides that would form the third and eighth extracellular segments according to the 4-MPM folding map.

On the other hand, the HA epitope inserted at site I (Ala264-Asn265) in the long hydrophilic loop yielded a fully functional protein, as judged by its ability to support yeast growth in submillimolar potassium (Fig. 4), i.e. growth equivalent to that supported by unmodified Trk2p expressed from the same plasmid and under the same conditions. Furthermore, the fluorescence image produced by HA at site I after reaction with primary and secondary antibodies was nearly identical to the corresponding native EGFP fluorescence image, as demonstrated in Fig. 5. Again, the antibody staining was clear only with cells that had been permeabilized by detergent treatment (Fig. 5, compare middle and left panels). Background staining of unpermeabilized cells via mouse anti-HA IgG was more evident than background staining by rabbit anti-GFP IgG (Figs. 3 and 5, compare upper middle panels), but this was largely due to a slight reactivity of anti-HA antibody with native yeast surface proteins (Figs. 3 and 5, compare right panels).



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  		FIG. 4.
HA- and EGFP-tagged Trk2p supports low K+ growth just as well as wild-type Trk2p does. Conventional drop tests (at four dilutions; see "Experimental Procedures") were performed with yeast maintained in limiting potassium (left panel) or in abundant potassium (right panel). Shown is the standard expression in the trk1{Delta} trk2{Delta} strain HY483 of unmodified plasmid pYGW1 (columns 2), pYGW1 bearing the wild-type TRK2 gene (columns 1), and the same plasmid bearing TRK2-HA-EGFP (columns 3).

 



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  		FIG. 5.
Anti-HA antibody decorates Trk2p-HA-EGFP only in permeabilized cells. The general configuration of the experiment and the display are similar to those described for Fig. 3, but using TRITC-conjugated anti-HA antibody. The important inference is that the HA epitope, inserted into the long hydrophilic loop of Trk2p, must reside at the inner surface of the yeast plasma membrane. The detectable background staining of fixed-only cells (upper middle panel) resulted largely from the slight reactivity of anti-HA antibody with native yeast surface proteins (cf. right panels).

 
The essential conclusion here is that the long hydrophilic loop tagged by the HA epitope does indeed lie at the intracellular surface of the yeast plasma membrane, as predicted by the 4-MPM map (K+-channel analog) of the TRK proteins and contradicting the 12-TM map.

Confirmation by Immunoelectron Microscopy—A potential caveat in the above argument is that proteins or even hydrophilic segments of proteins could, in principle, be folded so that particular epitopes would not be exposed for antibody binding without prior denaturation, regardless of which side of the plasma membrane actually bears the protein segment. This problem can be circumvented by analysis of antibody-complexed protein exposed in sliced cells that have not been treated with detergent. However, such an approach requires much higher spatial resolution than is available with the light microscope. Contrast labeling appropriate for electron microscopy studies was produced by protein A-gold acting as a secondary (EGFP) or tertiary (HA) "antibody." Transmission electron microscopy following freeze sectioning and uranyl acetate staining proved more satisfactory than the usual procedures for osmium fixation.

Typical distributions of gold-labeled HA inserted at site I in the long hydrophilic loop are shown in Fig. 6 (A and B). Throughout all micrographs (~40 from five different cell preparations), the gold particles lay preponderantly inside the surface membrane. In detailed counts, 82% of all particles were located just inside the plasma membrane, with the remainder scattered outside. Because the theoretical length of tether between the membrane-resident protein and each gold particle (via protein A plus secondary and primary antibodies) exceeded 150 Å, the observed separation between the inner leaflet of the membrane and the majority of gold particles was not surprising, nor was the fact that occasional particles appeared to be extracellular given that reactions with antibodies and protein A needed to be carried out after the material had been sectioned. Similar distributions of particles were observed with anti-GFP antibody and protein A-gold, as demonstrated in Fig. 6C (just above the white carets), although a much lower density of labeling was achieved on those sections.

Complementary Results—Both the fluorescence and immunoelectron microscopy yielded additional results (not discussed above) that will be useful in functional characterization of the TRK proteins as expressed from plasmids. These deal with three topics: (a) the nature of the punctate distribution of fluorescence at the cell surface, (b) the nature of intracellular clusters, and (c) a simple method to suppress the latter.

The punctate distribution of surface-localized fluorescence (Figs. 2, 3, and 5) is strongly reminiscent of protein associations with proteolipid rafts as described for yeast (35-37). We therefore attempted to determine whether tagged Trk2p would also satisfy other criteria for raft-like associations and explicitly whether specific antibody-reacting material in yeast membranes is resistant to extraction by Triton X-100, as originally described for protein anchoring by membrane glycolipids (38). The results, as shown in Fig. 7, were completely clear-cut: no antibody-positive material was retained in the high speed supernatant from broken cells by 2% Triton X-100 (SN, middle lane), whereas antigen in the low speed supernatant (Total, left lane) was readily pelleted and solubilized by 1% SDS (Pellet, right lane). By this standard criterion, yeast Trk2p does indeed organize into rafts within the membrane.

It is unlikely, however, that all of the fluorescent spots observed along the yeast plasma membrane in Figs. 2, 3, and 5 represent simple lipid rafts. Such a caveat is required because some electron micrographs from cells expressing plasmid-borne TRK2-HA-GFP revealed patches of gold-tagged cortical endoplasmic reticulum (ER) lying next to the plasma membrane, as demonstrated in Fig. 8C. Although the predominant image was of essentially linear (planar) arrays of gold particles nearly uniformly spaced along the inner surface of the plasma membrane, as shown in Fig. 6 (A and B), "short stacks" of gold-labeled ER membranes sometimes did confuse the image, especially in sections with conspicuous cytoplasmic membranous clusters (Fig. 8B). Thus, a fraction of the observed fluorescent spots likely represent not proteolipid rafts in the plasma membrane, but stacks of cortical ER.

Electron micrographs such as those in Fig. 8 (A and B) also clarified the nature of clumped cytoplasmic fluorescence, which could result from plasmid-borne expression of TRK2-HA-GFP (Figs. 2, 3, and 5); these spots proved to be jumbles of antibody-reactive membranes strongly resembling vesicular-tubular clusters (VTCs) of the ER, such as have been described in yeast and elsewhere under various conditions of stress (39, 40). Although VTCs are usually regarded as intermediates between the ER and the Golgi network, the actual station of the clusters observed here is not known. They could be either transient deposits antecedent to proteolysis and recycling of amino acids or ER "holding tanks," giving the associated proteins extra time to fold correctly.

Probably the most useful complementary result emerged from efforts to reduce the fraction of plasmid-expressed Trk2p that lodged in VTCs. Many neutral osmolytes such as glycine betaine, trehalose, and glycerol serve as chemical chaperones and have been found to assist protein folding in a variety of microorganisms, including yeast (41-44), under various conditions of stress. A simple expedient was therefore tested: growing yeast containing pYGW1-TRK-HA-EGFP in the usual YP-Gal medium, but supplemented with 5 or 10% glycerol. The results were dramatic and are illustrated in Fig. 9, with a false color representation of fluorescence intensity. Again, as in the experiment in Fig. 5, fluorescent (secondary) antibody to the HA epitope reached very few sites without prior detergent permeabilization of the cell membranes (upper panels), but permeabilized cells fluoresced intensely: predominantly at the plasma membrane of cells grown in 10% glycerol (lower right panel), but at progressively brighter and more numerous intracellular sites in 5% to 0 glycerol.



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  		FIG. 9.
Suppression of intracellular deposition of HA-tagged Trk2p by glycerol added to YPGal medium. Shown is the false color representation of intensities, as illustrated in the 8-bit color bar. Upper panels, cells fixed but not detergent-treated; lower panels, cells fixed and then treated with 0.1% Triton X-100. As described for Fig. 5, both membrane-bound Trk2p-HA and intracellularly aggregated Trk2p-HA were substantially accessible to the antibodies only in detergent-permeabilized cells. Glycerol above 5% (v/v) progressively abolished cytoplasmic fluorescence and VTCs, with negligible effects on the overall growth of yeast in up to 10% glycerol.

 
For the sake of maintaining yeast plasma membranes in as nearly "normal" conditions as possible in topological studies of Trk2p, compatible with adequate expression levels, we decided not to use glycerol growth for the main fluorescence measurements. However, some expected future applications of this proofed technique are listed under "Discussion."


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Comparative Analysis—As pointed out by several research groups (15, 16, 18), sequence comparisons between these proteins and bacterial potassium channels seem to identify, in all TRK proteins, four re-entry loops (P-loops), each containing a critical glycine residue that keys the signature GYG elements of K+-channel selectivity filters. About four dozen proteins are now known in the TRK family, and alignment of their putative P-loops is shown in Fig. 10. The five residues corresponding to the K+-channel selectivity filter are boxed, revealing many variants of the K+-channel signature triplet: most commonly GLN(T/S) or GFN(T/S). The glycine residue itself is almost fully conserved, exceptions presently found only in the first P-loop (Pa) of several plant TRK proteins, where a serine residue is substituted.



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  		FIG. 10.
Alignment of the four P-loops in the currently known TRK family proteins/genes. Separate consensus sequences are shown in the top line in each of the three groups (fungal, plant, and bacterial), and deviations from the consensus sequences are displayed below for 18 fungal proteins, 8 higher plant proteins, and 22 bacterial proteins. Total deviations from the consensus sequences, among the 76 residues shown for each species, are listed on the right. The five amino acids corresponding to the selectivity filter in KcsA are boxed; the K+-channel critical glycine is highlighted in gray. Note that the latter is conserved everywhere, except in Pa of several plant proteins. The fungal species are as follows: S. cerevisiae (Sc.), Saccharomyces bayanus (Sb.), Candida albicans (Ca.), Kluyveromyces lactis (Kl.), Neurospora crassa (Nc.), Debaryomyces occidentalis (Do.), Podospora anserina (Pa.), Aspergillus nidulans (An), Magnaporthe grisea (Mg), Fusarium graminearum (Fg), and Schizosaccharomyces pombe (Sp.). The higher plant species are as follows: Mesembryanthemum crystallinum (Mc.), Eucalyptus camaldulensis (Ec.), A. thaliana (At.), Oryza sativa (Os.), and T. aestivum (Ta.). The bacterial species are as follows: Thermoanaerobacter tengcongensis (Tt.), Desulfovibrio desulfuricans (Dd), Vibrio alginolyticus (Va.), Vibrio parahaemolyticus (Vp.), Vibrio vulnificus (Vv), Fusobacterium nucleatum (Fn.), Aquifex aeolicus (Aa.), Bacillus cereus (Bc.), Treponema pallidum (Tp), Synechococcus sp. (Ss), Desulfitobacterium hafniense (Dh), Shewanella oneidensis (So.), Listeria innocua (Li.), Borrelia burgdorferi (Bb.), Prochlorococcus marinus (Pm.), Streptococcus pneumoniae (Stp.), Deinococcus radiodurans (Dr.), Streptococcus agalactiae (Sta), Bacillus anthracis (Ba.), Mycoplasma pneumoniae (Mp.), Enterococcus faecalis (Ef.), and Mycoplasma pulmonis (Mpu.). Most fungi appear to possess two or more variant Trk sequences. Additional partial sequences are on file at www.genome.wi.mit.edu/annotation/fungi/fgi/index.html for Fusarium, Magnaporthe, Cryptococcus, Ustilago, and Coprinus.

 
Detailed structural modeling of the TRK proteins by overlay upon the x-ray-deduced structure of the KcsA channel (20) requires many deviations, due both to lack of precise symmetry among the pore loops and to numerous and sometimes extended insertions. In consequence, the expected pore or ion pathway through the TRK proteins would be much less regular than that defined for bona fide K+-channels (21).

Cytochemical evidence concerning folding of TRK family proteins began with a study of the wheat Triticum aestivum protein HKT1 tagged with the V5 epitope replacing a 16-amino acid span within the 60-amino acid hydrophilic segment that corresponds to long hydrophilic loop in the yeast Trk proteins. The tagged gene was expressed in S. cerevisiae, and the epitope was found to be accessible to extracellular antibodies in fixed but unpermeabilized cells (45). The hydrophilic loop was thus projected to lie extracellularly, counterindicating the 4-MPM model. Exactly the opposite conclusion was reached by Kato et al. (46) with the homologous HKT1 protein from Arabidopsis thaliana, which was tagged with three different markers: the PhoA protein (alkaline phosphatase) from E. coli, the FLAG epitope, and several synthetic glycosylation sites. The sidedness of these markers was assessed after expression either in E. coli or in a cultured mammalian cell line (HEK293), and all measurements conformed to the 4-MPM model.

The first important functional evidence was that of Tholema et al. (13) from site-directed mutagenesis of the KtrB protein, which mediates high affinity uptake of potassium in Vibrio alginolyticus. Replacement of Gly290 in the selectivity segment of the postulated third P-loop had drastic effects on transporter function; Ser290 and Asp290 produced nonfunctional enzymes, although the actual presence of normal amounts of protein in the Vibrio membrane was not verified. On the other hand, Ala290 produced about a 40% decrease in maximal transport velocity, but increased the apparent K0.5 for potassium by >30-fold (50 µM to 1.7 mM). Furthermore, functioning of the wild-type KtrB protein depended strongly upon extracellular sodium, and the Ala290 mutant displayed an even larger increase in the K0.5 for Na+ (40 µM to 6 mM).

These experiments have since been repeated, with variations, on homologous plant and fungal proteins. The A. thaliana HKT1 protein, with Ser68 in place of the signature glycine, and the HKT1 homolog in wheat (T. aestivum), which retains the glycine (Gly91), make an interesting comparison. A. thaliana wild-type HKT1 appears to be an Na+-Na+ transporter (47), whereas T. aestivum wild-type HKT1 seems to couple potassium transport with sodium influx (9, 12). But most importantly, the S68G switch in A. thaliana HKT1 and the G91S switch in T. aestivum HKT1 completely interchange the two phenotypes (48). In yeast Trk1p, extensive mutagenesis of the proposed eighth transmembrane helix, which should half-bracket the fourth putative P-loop, has demonstrated impressive changes in transport affinity due to modifications of charge along the helix, particularly at native Lys1147, Lys1158, and adjacent neutral residues (49). All of these functional changes are sensibly related to the 4-MPM map of Trk2p, but only indifferently related to the 12-TM map.

Folding of S. cerevisiae Trk2p Expressed in Its Native Membrane—The present topological study of Saccharomyces Trk2p necessarily focused on its long hydrophilic loop, which all evidence (both immunochemical and immunoelectron microscopic) located at the intracellular surface of the yeast plasma membrane. This result is fully compatible with the 4-MPM map of the protein (channel-like model) (21) and incompatible with the 12-TM model. Furthermore, the confirmed intracellular location of the C terminus and an even number of membrane-spanning segments (in both models) require the N terminus to be intracellular. This deduction implies also that only two (an even number of) bona fide transmembrane segments can exist ahead of the epitope-tagged loop, again supporting the 4-MPM model and contradicting the 12-TM model.

This localization of the long hydrophilic loop in yeast Trk2p conflicts directly with the finding of Liu et al. (45) concerning the corresponding loop in T. aestivum HKT1. At least three explanations can be imagined for this discrepancy. (i) T. aestivum HKT1 and Trk2p may actually fold differently in their native states; this is considered unlikely in view of the findings of Kato et al. (46) on A. thaliana HKT1. (ii) T. aestivum HKT1 heterologously expressed in yeast is in fact mistargeted, albeit not sufficiently to block all potassium transport by TRK-negative strains of Saccharomyces. (iii) The fluorescence technique applied to T. aestivum HKT1 went awry. An additional indication of this possibility lies in the one actual image of T. aestivum HKT1 distribution that documents the fluorescence report (see Fig. 5 of Ref. 45): the fluorescence intensity was remarkably uniform over the diameter of each cell, rather than even slightly concentrated at the edge, where it should appear amplified by a grazing optical section of the membrane. The actual image, then, suggests a cytoplasmic (not membrane) location for the expressed protein— again, mistargeting.

Melding of Carrier and Channel Concepts—The special interest in the fungal Trk proteins and their homologs in bacteria and plants started in their sequence homology to bacterial potassium channels (15-18). That, in turn, led to atomic-scale modeling and detailed prediction of KcsA-like folding of the TRK proteins (21). The emerging demonstrations, including this one, of the accuracy of this prediction firmly launch the notion that membrane proteins "originally" designed as ion channels can be modified to function as carriers, in the classical sense of primary or secondary active transporters. This notion merges comfortably with many other observations and ideas about membrane transport, e.g. that clusters of membrane-spanning {alpha}-helices characterize the vast majority of transport proteins, regardless of their operational mode (viz. channels, cotransporters, antiporters, ATP-driven pumps, gradient-driven ATP synthases, photon-driven pumps, electron-driven pumps, etc.); that most ion channels in eukaryotic systems are bounded by one or more "gates" that control access to the actual pore structure; and that bona fide ion pumps can also be switched on and off by gating mechanisms not directly related to the actual passage of substrates through the membrane (50, 51).

The first mechanical analog of an ion pump was a V-shaped rocking valve (52), which was easily transformed into a double-gated channel and even into a peristaltic channel (53, 54) and whose picture has turned out to be uncannily similar to alternating access models of the recently crystallized lactose-H+ cotransporter LacY and phosphate/glycerol phosphate exchanger GlpT from E. coli (30, 55). These are certified 12-TM transporters. It is probably safe to say that channel-like structures are an intrinsic feature of all biological membrane transport proteins and that all physiological carrier proteins can fall or be pushed into channel-mode functioning (56, 57). This conclusion is particularly germane to the TRK proteins because patch-clamp studies in situ have shown that chloride loading of yeast cells evokes relatively huge inward currents (outward flux) of chloride ions via both Trk1p and Trk2p,2 i.e. potassium carriers can also function as chloride channels. Indeed, a very similar phenomenon has already been reported for neurogenic amine/neurotransmitter transporters heterologously expressed in Xenopus oocytes (59, 60), which have a chloride-rich cytoplasm (61). The folding arrangement of these proteins is not yet clearly understood, but eight transmembrane helices, at least two re-entry loops, and at least one long hydrophilic loop are conspicuous features (62, 63).

Additional Implications—The combination of fluorescence microscopy, electron microscopy, and lipid extraction analysis has indicated two sources for aggregation of yeast proteins at/near the plasma membrane: conventional proteolipid rafts (Fig. 7) and stacks of cortical ER (Fig. 8). Identification of the former adds Trk2p, and presumably also Trk1p, to the growing list of proteins (in yeast and elsewhere) that are so organized (36, 64-66). But a remarkable feature of the published information on yeast is the near absence of ultrastructural evidence. Given the fact that cortical ER lying directly inside the plasma membrane can mimic the surface appearance of rafted proteins, as suggested by Fig. 8C, some caution is called for in the interpretation of fluorescence images. (It must be noted, however, that Harder et al. (65) have produced detailed electron microscopic evidence consistent with the formation of proteolipid rafts in BHK cultured cells.)

From cytological appearances, the tangled arrays of intracellular membranes that can be produced by plasmid-borne expression of TRK2 have been designated vesicular-tubular clusters, after the original descriptions on cultured mammalian cells (39, 67, 68) and related work on yeast (40, 69, 70). These clusters are far more random in both internal structure and cytoplasmic placement than typical karmellae, which have been described for overexpression of some other membrane proteins in yeast (58, 71). Proper karmellae are multilayered structures usually located perinuclearly and have been suggested to be storage organelles proliferating from ER membranes (58), whereas VTCs have been identified as transient intermediates between the ER and the Golgi network. Functions for the present VTC-like structures in yeast, perhaps as relay stations either for protein degradation or for enhanced folding, remain to be determined.

However, the fact that these VTC-like structures can be dispelled by growth of yeast cells in 5-10% glycerol, which is well known as a chemical chaperone, suggests that misfolded or mistargeted protein can be functionally rescued. This conclusion could have great practical application, particularly in the study of heterologously expressed membrane proteins. As a general rule, plant and fungal membrane proteins readily express with nearly normal function in yeast plasma membranes, but animal membrane proteins rarely do, despite abundant evidence that their encoding genes are transcribed. The reasons for this practical incompatibility between yeast membranes and animal membrane proteins are not understood, but improper folding with subsequent intracellular aggregation and/or degradation is high on the list of possibilities. We are therefore undertaking the testing of the effect of glycerol growth on the functionality of several ion-channel proteins encoded on yeast expression plasmids. This same issue is also being explored in several other laboratories, particularly that of Figler et al. (42), who have described greatly enhanced functioning of human MDR1 (P-glycoprotein) expressed in Saccharomyces.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Research Grant GM-60696 (to C. L. S.) and a Yale Medical School James Hudson Brown-Alexander Brown Coxe fellowship (to G.-F. Z.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Dept. of Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06520. Tel.: 203-785-4478; Fax: 203-785-5535; E-mail: clifford.slayman{at}yale.edu.

1 The abbreviations used are: PBS, phosphate-buffered saline; HA, hemagglutinin; GFP, green fluorescent protein; EGFP, enhanced green fluorescent protein; TRITC, tetramethylrhodamine isothiocyanate; ER, endoplasmic reticulum; VTC, vesicular-tubular cluster. Back

2 T. Kuroda, H. Bihler, E. Bashi, C. L. Slayman, and A. Rivetta, submitted for publication. Back


   ACKNOWLEDGMENTS
 
We are indebted to the Yale Medical School Keck Biotechnology Resource Laboratory for synthesis of oligonucleotides (John Flory and Joseph DeLuca) and for sequence analysis (Karl Hager and Emily Hilton) and to the Center for Cell and Molecular Imaging (Philippe Male and Kimberly Zichichi) for assistance with fluorescence and confocal microscopy and for preparation of electron microscopic sections. We are also indebted to Esther Bashi for assistance and advice throughout the experiments and to Michael Caplan for use of the Zeiss Axiophot fluorescence microscope. R. F. Gaber generously supplied the TRK deletion strain of yeast. Peter Novick, A. Brett Mason, and Carolyn W. Slayman were imaginative and helpful critics of the manuscript, and Dr. Mason advised and assisted with the lipid extraction experiments. We are indebted to H. Robert Guy (National Institutes of Health) for the stimulus to begin this project and for many helpful suggestions and to Dr. Guy and Jay Dunlap (Dartmouth College) for assistance in tracking down the more than four dozen TRK gene sequences.



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 DISCUSSION
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