N-1 -Naphthylphthalamic Acid-Binding Protein an Integral Membrane

Plant Physiol. (1 996) 11 1 : 427-432

The N-1 -Naphthylphthalamic Acid-Binding Protein 1s an Integral Membrane Protein

Paul Bernasconi*, Bhavesh C. Patel, Jeff D. Reagan, and Mani V. Subramanian

Sandoz Agro Inc., Research Division, 975 California Avenue, Palo Alto, California 94304-1 104

N-1 -Naphthylphthalmic acid (NPA)-binding protein is a plasmalemma (PM) protein involved in the control of cellular auxin efflux. We re-evaluated the spatial relationship of this protein with the PM of zucchini (Cucurbifa pepo L.) hypocotyls. First, Triton X-114 partitioning indicated that the NPA-binding protein was more hydrophobic than most PM proteins. Second, the NPA-binding activity was found to be resistant to proteolytic digestion in membranes. Maximum concentrations of binding sites for NPA were virtually identical in untreated and proteinase K-treated PMs: 19.2 and 20.6 pmol [3H]NPA bound/mg protein, respectively. The insensitivity of the NPA-binding protein was not due to its presence inside tightly sealed vesicles or due to lack of protease activity in the conditions tested. This protein could be made sensitive to proteolytic degradation upon solubilization by 3-[(3-~holamidopropyi)dimethyiam- moniol-1 -propanesulfonate in the presence of sodium molybdate. Proteinase K treatment decreased the concentration of binding sites to 0.84 pmol L3H1NPA bound/mg protein from 9.2 for untreated, solubilized PM. Third, this activity could not be solubilized by chaotropic agents or sodium carbonate treatment of intact PM. This study indicates that the NPA-binding protein may be an integral membrane protein and contradicts previously reported findings that suggested that this protein was peripheral to the PM.

NPA is a synthetic phytotropin that severely stunts the growth of plants (Hoffman and Smith, 1949). Early physi- ological characterization of the NPA mode of action re- vealed that it reduced auxin transport in stems and roots (Morgan, 1964) and that the stunted growth and the inhi- bition of the gravitropic response were a consequence of NPA-induced auxin accumulation in the cells (Morgan, 1964). Although the physiology of plants treated with NPA has been intensely studied and the compound itself has been made into a commercial herbicide (Naptalam, Uni- royal, Middlebury, CT), surprisingly little is known about the biochemical aspects of its mode of action (for a review, see Lomax et al., 1995).

It is now well established that NPA specifically inhibits the efflux of auxin from the cytoplasm of the cell into the periplasm (Lomax et al., 1995). The availability of radiola- beled NPA allowed the characterization of a single, satu- rable NPA-binding activity located on the PM (Lembi et al., 1971; Muday et al., 1993).

A further understanding of the identity of the NPA- binding activity and the mechanism by which it regulates auxin efflux was attempted by several groups. One ap-

* Corresponding author; e-mail bernasconiQsandoz.com; fax 1-415-493-1073.

427

proach has been to search for the natural counterpart of NPA, and several candidates such as quercetin (Jacobs and Rubery, 1988), hydrangeic acid, or lunularic acid (Katekar et al., 1993) have been proposed. Another approach has been to investigate the NPA-binding transduction path- way. These studies have uncovered a potential role of Tyr kinase for the NPA-binding protein (Bernasconi, 1996), which possibly phosphorylates a small soluble protein (Morris et al., 1991). This phosphorylated protein would in turn regulate the activity of the auxin carrier.

Independently, it has been proposed that the NPA-binding protein is peripheral to the PM and interacts with actin filaments (Cox and Muday, 1994). The significance of such an association with respect to auxin efflux regulation is not yet clear. A peripheral location of the NPA-binding protein is surprising in several ways. First, the NPA-binding activity has been difficult to solubilize (Brunn et al., 1994), a property that is usually not associated with peripheral proteins. Second, and more important, several analogs of NPA have been prepared and a correlation has been found between the hydrophobicity of a compound and its ability to displace bound NPA (Katekar and Geissler, 1977; Brunn et al., 1994). This finding could not be explained if the binding protein were peripheral to the membrane, since the compound would partition in the membrane, becoming inaccessible to the binding protein. It is therefore important, at this point, to further refine the subcellular localization of the NPA-binding activity. With that intent, we undertook a study of the sensitivity of the NPA-binding protein to proteolysis in both intact PM and in solubilized fractions obtained using a nove1 solubilization protocol.

MATERIALS AND METHODS

Chemicals

NPA was from Chem Services (West Chester, PA). [2,3,4,5-3H]NPA (58 Ci/ mmol) was from American Radio- labeled Chemicals (St. Louis, MO). Proteinase K was from Life Technologies. Sodium molybdate was from Mallinck- rodt (Paris, KY). Dextran T500 was from Pharmacia-LKB. Ready Protein scintillation cocktail was from Beckman In- struments. The Micro BCA protein assay kit was from Pierce. Triton X-114 was obtained from Boehringer Mann- heim and precondensed as described by Bordier (1981) before use. A11 other chemicals were from Sigma.

Abbreviations: B,,,, maximum concentration of binding sites; Chaps, 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate; NPA, N-1-naphthylphthalamic acid; PM, plasmalemma.

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428 Bernasconi et al. Plant Physiol. Vol. 11 1, 1996

Plant Material and P M Preparation

Zucchini (Cucurbita pepo L.) seedlings were grown for 5 d at 28°C in the dark. PM was prepared from the upper 5 cm of the hypocotyls using the two-phase partitioning method described by Widell and Larsson (1981). The PM fraction obtained from 70 g of tissue was resuspended in 1 mL of PM buffer (250 mM SUC, 20 mM sodium citrate, 1 mM magnesium chloride, pH 5.3) to yield approximately 5 mg protein/mL (Pierce Micro BCA protein assay). Membranes were ali- quoted, frozen in liquid nitrogen, and stored at -80°C.

Protein Solubilization

Published solubilization methods were adapted for the material used (Jones et al., 1987; Haga et al., 1990). PM fractions were adjusted to 2.25 pg protein/ pL and incubated for 2 h at 4°C in PM buffer containing 0.6% Chaps, 10 mM sodium molybdate, 1 mM DTT, and 10% glycerol. Unsolubilized material was pelleted by a 45-min centrifugation at 100,OOOg. The supernatant, containing the solubilized fraction, was frozen in liquid nitrogen and stored at -80°C.

NPA-Binding Assays

NPA-binding assays were conducted using 10 to 30 pg PM protein/sample. Samples were incubated for 2 h at 4°C with 10 nM 13H]NPA in a volume of 200 pL of PM buffer. Nonspecific binding was determined in the presence of 10 p~ unlabeled NPA. Bound [3H]NPA was separated from free [3H]NPA by filtering over GF/B filters (Whatman) presoaked in 0.1% polyethylenimine (Bruns et al., 1983). The filters were washed with 5 mL of cold PM buffer and counted in 4 mL of Ready Protein cocktail. Saturation assays were conducted with concentrations of [3H]NPA ranging from O to 50 nM. B,,, and K , values were determined from the saturation curves by the program LIGAND (Munson and Rodbard, 1980). NPA binding in the solubilized material was conducted in a similar fashion but in the presence of 0.2% Chaps and 1 mM sodium molybdate in the PM buffer. In the cases in which a saturation curve was not determined, the experiments were performed at least three times and the average value of specific binding from a typical experiment was reported.

Triton X-114 Phase Partitioning

PM fractions (0.5-1 mg) were submitted to Triton X-114 partitioning (Pryde, 1986). The published method (Pryde and Phillips, 1986) was followed exactly with one excep- tion: the aqueous phase obtained after the SUC cushion separation was used directly without dialysis. As a consequence, a small amount of contamination by the detergent phase may be present. After determination of the protein content by the Micro BCA protein assay, NPA-binding assays were performed as described above using 10 pg protein/ assay.

Protease Treatments

Unless noted otherwise, proteinase K treatments were performed in PM buffer for 16 h at 4°C. The ratio of

proteinase K to PM protein was 1:2, with the final proteinase K concentration being 1 pg/ pL. Alternatively, pronase E and trypsin were also used under similar conditions. After protease treatment, the fractions were diluted in PM buffer and assayed for NPA binding as above and analyzed by SDS-PAGE.

SDS-PACE and Western Blot Analysis

SDS-PAGE and western blot analysis were performed according to published methods (Ausubel et al., 1995). Monoclonal anti-actin antibodies (Cox and Muday, 1994) were used at a 1:400 dilution according to the manufactur- er's instructions (ICN Pharmacologicals). Detection was performed using the ECL detection kit (Amersham Life Sciences) and Fuji medica1 x-ray film RX (Fisher Scientific).

RESULTS A N D DlSCUSSlON

NPA-Binding 1s Associated with the Purified P M Fraction

The two-phase partitioning protocol was used for the preparation of the PM fraction. An average of 6% of the total microsomal protein partitioned as PM, with the remaining protein partitioning as a mixture of subcellular membranes. Cyt c oxidase activity, a mitochondria marker (Hodges and Mills, 1988), was used as a marker. The al- most complete depletion of this activity in the PM fraction (more than 900/) was an indication of the small extent of contamination by other membranes. These results were comparable to those obtained with other plant tissues (Hodges and Mills, 1988). It is well established that the NPA-binding activity co-purifies with PM markers. In- deed, after the initial study by Lembi et al. (1971), NPA- binding activity has often been used as a marker of PM fractions (Chanson et al., 1984; Hodges and Mills, 1988). In our material, the specific activity of the NPA-binding protein was enriched 3-fold in the PM fraction when compared to the total membrane fraction (Table I). On average, the B,,, value for the NPA-binding protein increased from 6 to 8 pmol/mg protein in the total microsomal fraction to 19 to 24 pmol/mg protein in the PM fraction. A representative saturation curve for the NPA-binding activity in PM is shown in Figure 1A. These results are well in accordance with severa1 previous reports (Muday et al., 1993; Lomax et al., 1995).

Table 1. B,,, and Kd values of the NPA-binding activity Total microsomal fraction, two-phase partitioning-purified PM,

and Chaps/sodium molybdate-solubilized PM were used with or without overnight pretreatment at 4°C in the presence of 1 &pL proteinase K. The values given were calculated with the program LIGAND from saturation curves in the Dresence of O to 50 nM NPA.

~~

Fraction Treatment B,,, Kd

pmo//mg protein nM

Total membranes None 6.7 6.8 PM U ntreated 19.2 7.09

Proteinase K 20.6 8.23 Solubilized PM U ntreated 9.20 6.93

Proteinase K 0.84 6.32



N-1 -Naphthylphthalamic Acid-Binding Protein Is Integral to the Plasmalemma 429

A

B 3H NPA (nM)

0 10 20 30 40 50 60

3H NPA (nM)

Figure 1. NPA-binding activity in membrane-bound (A) and solubilized (B) PM fractions. The saturation curves were obtained as in Table I . The binding experiment was conducted with PM fractions incubated overnight at 4°C in the absence (O) or presence (O) of 1 &pL proteinase K.

NPA-Binding Activity Partitions in the Highly Hydrophobic Phase during Triton X-114 Partitioning

To give a first indication of the hydrophobicity of the NPA-binding protein, PM fractions were subjected to Tri- ton X-114 phase partitioning. Although this partitioning is not a solubilization protocol per se (Pryde, 1986), it allows an approximate determination of the hydrophobicity of a protein and has been successfully used in animal (Pryde and Phillips, 1986) and plant systems. A recent example is the resolution of the membrane localization of the fusicoccin receptor in Commelina communis tissues (Oecking et al., 1994). In our system, this protocol yielded three separated phases of increasing hydrophobicity: an aqueous phase, a detergent-rich phase, and a lipid-rich phase. The result of a representative experiment in which 500 pg of PM protein were used is given in Figure 2. About 50% of the initial protein was recovered in the three phases, with the majority of the protein found in the detergent-rich phase. The severa1 pellet washes and SUC gradient interphases were responsible for the protein loss. The NPA-binding activity partitioned preferentially in the lipid-rich phase, indicating that the NPA-binding protein was more hydrophobic than the majority of the PM protein (Pryde, 1986). Although some activity was found in the detergent-rich phase, none was detected in the aqueous phase. The partitioning pro- toco1 somewhat denatured the NPA-binding protein; therefore, the specific NPA-binding activity, even in the fraction enriched in this activity, was only about 30% of the binding in the starting material.

NPA-Binding Activity 1s lnsensitive to Proteolysis

Since the NPA-binding protein partitioned with the highly hydrophobic phase, it could be postulated that this protein is intrinsic to the PM and inaccessible to proteases. Incubation of PM fractions with proteases would therefore not affect NPA binding. Our investigations indicated that the NPA-binding activity is essentially insensitive to an overnight treatment in the presence of a large amount of proteinase K (1 pg proteinase K / 2 pg PM protein). The results reported in Figure 1A and Table I showed no change in B,,, or in K , between proteinase K-treated and untreated fractions. As a consequence, this treatment did not decrease the number of binding sites, and it did not alter the affinity for NPA of the binding sites. These findings contradicted the results of Sussman and Gardner (1980), showing that this activity was sensitive to trypsin digestion. This contradiction may be due to three differ- ences between our study and the published report. First, we benefitted from the development by Widell and Lars- son (1981) of the two-phase PM preparation system, allow- ing us to work with highly purified PM fractions as op- posed to crude microsomal preparations. Second, we could replace the column chromatography method for NPA- binding assays used by these authors with the filtration o? GF/B filters described by Bruns et al. (1983). This method allowed measurements of NPA binding, with nonspecific binding contributing 5% or less of the total binding. It also avoided the limitation due to the rapid off-rate of NPA from the binding protein (77 and 90% of the radioactivity released from the protein after 5 and 10 min, respectively; Patel et al., 1995). Third, it appears that the binding activity

.f 20 B k 15 2 3 10

3

'c

O

aqueous detergent rich lipid rich <- phase

Figure 2. Triton X-114 phase partitioning of PM proteins (top) and NPA-binding activity (bottom). The three fractions were obtained starting with 500 pg of PM protein. These fractions, reported by increasing hydrophobicity, corresponded to the ones described by Pryde and Phillips (1986). The results are given in percentage of recovered protein (top) and pmol I3H1NPA bound/mg protein of the corresponding phase (bottom). The protein was assayed using the Pierce Micro BCA protein assay kit. The NPA binding was measured on 10 pg of protein in the presence of 10 n M r3H1NPA. The experiment was repeated three times and a representative result is reported.



430 Bernasconi et al. Plant Physiol. Vol. 111, 1996

observed by Sussman and Gardner (1980) was very unsta-ble, since, even in the absence of a protease, a 20-minincubation at room temperature of their sample reducedthe NPA binding from 68,467 dpm/4 g fresh weight equiv-alent (Sussman and Gardner, 1980, table II) to 584 dpm/1.1g fresh weight equivalent (Sussman and Gardner, 1980,table IV). Hence, their analysis of trypsin degradation wasconducted on a membrane fraction having lost more than97% of its NPA-binding activity. Our results also contra-dicted the findings of Cox and Muday (1994) that indicatedthat the NPA binding was peripheral to the PM and locatedon the cytoplasmic side of the membrane. Hence, we in-vestigated alternative explanations that would reconcileour findings with their hypothesis.

NPA-Binding Protein Was Not Protected because ofTightly Sealed Vesicles

PM preparation by the two-phase partitioning methodhas been shown to yield sealed vesicles with the periplas-mic side of the membrane on the inside of the vesicle. Onepossibility was that proteinase K was not reaching theNPA-binding protein because it could not cross the tightlysealed membrane vesicle. This was ruled out by two ex-periments. First, PM vesicles were subjected to threerounds of freezing and thawing. Second, PM vesicles weresubjected to three 10-s bursts of sonication. These twomethods decreased the integrity of the membrane vesicles(Hertel et al., 1983). After these treatments, the fractionswere incubated with proteinase K and assayed for NPAbinding. No increase in sensitivity toward the protease wasobserved after sonication or freeze/thawing (data notshown, but similar to the Bmax and Kd values reported forPM in Table I).

In the Conditions Tested, Proteinase K Was Active

Another possible explanation for the insensitivity of theNPA-binding protein to digestion by proteinase K wassimply the lack of activity of the protease in the conditionstested. Proteinase K is stable between pH 4 and 12.5 and attemperatures up to 65°C. Our experimental design usedthis protease at pH 5.3 and 4°C. The choice of these condi-tions was dictated by the need to preserve the NPA-bind-ing activity. We demonstrated that proteinase K was suf-ficiently active in these conditions in three ways. Figure 3shows an SDS-PAGE analysis of the fractions subjected toan overnight incubation in the absence or presence of pro-teinase K. The majority of the proteins were degraded bythe protease, resulting in an accumulation of silver-stain-reactive material of small molecular mass. The seconddemonstration of proteinase K effectiveness utilized actin,a cytoplasmic protein associated with the membrane, as amarker for peripheral proteins. By immunoblotting, actinwas detected in total microsomal preparations, and a smallfraction, estimated at 5% of the total immunoreactivity,was found in the PM preparation (Fig. 4). This result was inaccordance with the findings reported by Sonesson andWidell (1993). Upon proteinase K treatment, no actin wasdetected in the sample, indicating that the protease effec-

Enz. PM Sol.K E T S t d C K E T C K B

_97.4

_66.2

_45.0

_31.0

_2I.5

Figure 3. Effect of protease digestion (overnight at 4°C) on NPA-binding activity and protein pattern in PM fractions. The NPA-bind-ing activity was assayed in duplicate in the presence of 10 nM[3H|NPA. A, For each sample, the average specific binding activity isgiven in pmol [3H]NPA bound/mg PM protein. B, For each treatment,the corresponding SDS-PAGE analysis is shown. Identical amounts ofprotein (10 /xg) were used for both analyses. Enz., Enzymes used inthe digestion; PM, membrane-bound fractions; Sol., solubilized frac-tion; Std, standards; C, control (no protease); T, trypsin; K, proteinaseK; E, pronase E. The molecular masses are given in kD on the right.Silver staining was performed to reveal the proteins remaining in thedigested samples and resulted in an overstaining of the undigestedsamples. The experiment was repeated three times and a represen-tative result is reported.

tively degraded this protein. The third demonstration ofproteinase K effectiveness was obtained by conducting theprotease digestion at 37°C for 1 h instead of the overnightincubation in the cold. Although this treatment resulted inabout 20% inactivation of the binding activity in the ab-sence of the protease, no further decrease in activity wasdetected in the presence of proteinase K (data not shown).

Pronase E and Trypsin Could Substitute for Proteinase K

To further demonstrate that the NPA-binding proteinwas insensitive to proteolytic digestion, the same exper-iments were repeated with pronase E and trypsin insteadof proteinase K. The results are reported in Figure 3.Likewise, incubation for 1 h at 37°C did not decreaseNPA-binding activity in PM (data not shown). Our tryp-sin treatment could not be directly compared to the one www.plantphysiol.orgon January 3, 2019 - Published by Downloaded from

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N-1-Naphthylphthalamic Acid-Binding Protein Is Integral to the Plasmalemma 431

o-

6-MEo 4-8ex

2-

A o-

B

1 1 — 11 1 1 1

•

TM PM SolFigure 4. Effect of proteinase K treatment on NPA-binding activity(A) and actin (B and C). PM fractions before (PM) and after solubili-zation (Sol) were incubated overnight at 4°C in the absence (-) orpresence (+) of 1 ng//AL proteinase K and compared to total micro-somes (TM). The immunoreactive actin signal was determined usingthe ECL reagent in a 5-min (B) or 10-s (C) exposure to x-ray film.

presented by Sussman and Gardner (1980), since in theirreport there was no indication of the amount of trypsinused and the ratio between trypsin and plant protein. Itwas reasonable to assume that the trypsin digestion inour protocol was at least as complete as theirs, since inour material the incubation was for 1 h at 37°C (insteadof 20 min at room temperature) using a high ratio oftrypsin versus PM protein. It should also be noted that,based on the SDS-PAGE analysis, trypsin was the leasteffective of the three proteases tested and a more com-plete digestion was obtained with pronase E and pro-teinase K (Fig. 3B).

NPA-Binding Activity Could Be Solubilized byChaps and Molybdate

Our data so far suggested that the NPA-binding proteinmay be protected against proteolytic digestion by the PM. Ifthat was indeed the case, a solubilized protein should be fullysusceptible to protease digestion. Before addressing this ques-tion, it was necessary to improve the existing solubilizationmethods for the NPA-binding protein. We evolved a protocolusing Chaps and molybdate that consistently solubilized 20 to25% of the NPA-binding activity from the PM fraction. Arepresentative saturation curve obtained with the solubilizedfraction is given in Figure IB. The solubilization did not affectthe affinity for NPA, since the observed Kd was similar in

both membrane-bound and solubilized fractions (Table I).The variation of the Bmax was probably due to the differentialsolubilization of certain proteins versus others, affecting thenumber of sites per unit of protein. The role played by mo-lybdate in this protocol was not fully understood, but thesame effect on solubilization yields was observed only withtungstate, albeit to a lesser extent. It can be argued that thision stabilizes the activity by inhibiting possible phosphatasesor other protein-modifying activities (Manchester et al, 1987).This protocol differed from the solubilization methods re-ported by three groups (Sussman and Gardner, 1980; Jacobsand Gilbert, 1983; Cox and Muday, 1994). The common fea-ture of these published reports is the use of Triton X-100 inconcentrations ranging from 0.1 to 2.5%. Sussman and Gard-ner (1980) described a 32% (their table I) or 56% (their table II)solubilization of the activity from the total microsomal frac-tion in the presence of 1% Triton X-100. No solubilizationyield was given in the other two publications, but it appearedto be much lower than the one obtained by Sussman andGardner (1980).

In our experimental system, no solubilized NPA-bindingactivity was recovered irrespective of the amount of TritonX-100 used. The apparent discrepancy may come from thedifferences in the analytical method used for assessingsolubilization. In our case, solubilized material was definedas one that could not be pelleted during a 45-min centrif-ugation at 100,000g (Hjelmeland and Chrambach, 1984). Adifferent criterion was used in the Sussman and Gardnerand Cox and Muday articles, in which solubilized materialwas defined as the supernatant of a 30-min centrifugationat 40,000£. The 100,000^ criterion was applied by Jacobsand Gilbert (1983) in their analysis of solubilized NPA-binding protein from pea. In this latter case, the solubilizedactivity was low and could not be compared directly withour findings (466 dpm/assay in table I or 752 dpm/assay intable II; Jacobs and Gilbert, 1983). The same low activitywas recovered by Thein and Michalke (1988) in their solu-bilization protocol involving 1% Chaps. This result wasconsistent with our preliminary studies using 0.4% Chapswithout sodium molybdate, yielding about 2.3% solubi-lized activity. Another solubilization method, reported byCox and Muday (1994), utilized 0.4 M potassium iodide or1 M sodium carbonate with solubilization yields of 80%,leading these authors to propose a peripheral location forthe NPA-binding protein. Attempts to replicate these re-sults with our material were unsuccessful. In the case of thecarbonate treatment, we found that addition of 1 M sodiumcarbonate to the reaction mixture increased the pH to 11and irreversibly denatured the NPA-binding protein. As aconsequence, the activity was not recovered in either thepellet or the supernatant. Finally, the tight interaction ofthe NPA-binding protein with actin was proposed by Coxand Muday (1994) as a possible reason for the low yield ofsolubilization of the activity by detergents. In our studies,using nonsolubilized PM, digestion of actin by proteinaseK (demonstrated by western blot analysis; see Fig. 4) didnot release any NPA-binding activity. This activity wasquantitatively recovered in the pellet after centrifugation at100,000# for 45 min.



432 Bernasconi et al. Plant Physiol. Vol. 11 1, 1996

Solubilized NPA-Binding Protein Was Susceptible to Protease Treatment

The solubilized fractions containing the NPA-binding activity were subjected to the protease treatments. A s reported i n Figure 18 and Table I, a n overnight incubation a t 4°C with proteinase K completely abolished NPA-binding activity i n these fractions. Similar results were obtained when the proteinase K w a s replaced by trypsin and pronase E or when the proteinase K digestion w a s performed for 1 h at 37°C (data not shown). The extent of the digestion w a s demonstrated by SDS-PAGE analysis (Fig. 38) and immunoblotting (Fig. 4, B and C). Since only 23% of the activity w a s solubilized i n this protocol, we also investi- gated the remaining activity that pelleted after treatment with Chaps a n d molybdate. The nonsolubilized protein w a s also readily digested by proteinase K and no activity was observed (data not shown).

Taken together, these results suggest that the NPA-binding protein may be hydrophobic and integral to the PM. This will be of importance for evaluating potential candidates for this protein isolated by molecular cloning methods. Although the present data directly contradict Cox and Muday’s (1994) work that found the NPA-binding protein to be peripherally associated with the PM, it does not rule out a possible interaction between a domain of this protein exposed to the cytoplasm and actin filaments. The findings i n the present report are in accordance with the correlation between the hydrophobicity of a compound and its effectiveness i n displacing N P A (Katekar and Geissler, 1977). They also raise the interesting possibility that, since the NPA-binding protein is mostly hydrophobic and N P A or NPA-displacing ligands are hydrophobic, the NPA-binding site may reside inside the membrane a n d be accessible to the ligand only after its partitioning i n the membrane.

Received December 18, 1995; accepted March 5, 1996. Copyright Clearance Center: 0032-0889 / 96/ 111 / 0427/ 06

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N-1 -Naphthylphthalamic Acid-Binding Protein an Integral Membrane

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