5
Proc. Natl. Acad. Sci. USA Vol. 81, pp. 4804-4808, August 1984 Cell Biology Isoelectric focusing of plant cell membranes (pI determinations/Fucus/Golgi/plasma membrane/polar transport) LAWRENCE R. GRIFFING* AND RALPH S. QUATRANOt Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331 Communicated by Winslow R. Briggs, February 29, 1984 ABSTRACT An isoelectric focusing method is described that discriminates plant cell organelle populations on the basis of surface charge. The isoelectric points (pI values) of the Gol- gi apparatus, the mitochondria, and putative plasma mem- brane from etiolated pea stem cells are reported. The pI of the pea Golgi apparatus is similar to that of the 35SO4-labeled membranes of developing Fucus embryos. The pI of Fucus 35SO4-labeled membranes depends on whether the membranes are being polarly transported to the growing tip or randomly transported to the entire periphery of the zygote. Those sub- ject to polar tip transport have a significantly greater negative surface charge than those being randomly transported. The implication of this result toward an understanding of the local- ization mechanism is discussed. The method is also capable of subfractionating glucan synthetase H-containing membranes (putative plasma membrane) from peas. The pI of putative plasma membrane from a pea stem homogenate is similar to the pI of the plasma membrane of whole protoplasts of Cathar- anthus leaf cells. Isoelectric focusing appears to be a useful technique to discriminate membranes and, hence, to provide new information and approaches to study cellular and devel- opmental phenomena. Isoelectric focusing (IEF) is used extensively to purify pro- teins, usually employing a polyacrylamide gel as a support medium (1). IEF can also be used to purify substances of very high molecular weight if a density gradient is used as an anticonvection medium. The advantages of a density gradi- ent for high molecular weight samples are rapid recovery of samples and short focusing time. Several IEF columns of low volume have been designed for a variety of purposes (2- 6). The low-volume density gradient column used in this study is similar to that described by Sherbet (7) for studies on animal cell surface charge. The operational characteris- tics are similar to the IEF-M3 column he described (7). The pI of most plant and animal organelles and membrane fractions is not known. The phase partition method of pI de- termination has been used to study the surface charge on the membranes of the chloroplast (8). A combination of phase- partition cross-point determination and IEF in a sucrose gra- dient has been used to establish the pI of animal cell mito- chondria (9). Variation in membrane composition make it likely that the surface differences of organelles will be reflected as a differ- ence in their pI values. In our studies on the polarity of zy- gotes of the brown alga Fucus, we attempted to apply IEF as a method to demonstrate a difference in the pI of vesicle fractions subject to polar or nonpolar transport. We report here details of this procedure and the results from the Fucus experiment. In addition, we demonstrate how the technique is applied to the determination of the pI of organelles and membrane fractions as well as its potential use for prepara- tive separation and isolation of subcellular particles and whole plant protoplasts. MATERIALS AND METHODS Plants and Chemicals. Pea seeds (Pisum sativum L. var. Alaska) were from Burpee Seed (Riverside, CA). Pea seeds were sown in vermiculite and watered every other day with tap water. They were grown in the dark at room temperature and all manipulations were done under dim red light. After 8 days, the epicotyls were harvested and an 8-mm segment was cut from 3 mm below the apical hook after they had been abraided with emery (aluminum oxide 400, Edmund Scien- tific, Barrington, NJ). Fucus distichus L. receptacles were collected from Yaquina Head (Newport, OR), and treated as described for the release and synchronous development of zygotes (10). Catharanthus roseus plants were made avail- able by F. Constabel (Plant Biotechnology Institute, Nation- al Research Council, Saskatoon, Sk., Canada). Cytochrome c (type III), dithioerythritol, UDPglucose and cellobiose were from Sigma. Biolyte carrier ampholytes 3/10, 3/5, 4/6, and 7/9 were from Bio-Rad. [1-3H]Glucose (specific activity, 4.5 Ci/mmol; 1 Ci = 37 GBq) and [14C]UDPglucose (specific activity, 275 mCi/mmol) were from ICN; H235504 (specific activity, 150 mCi/mmol) was from New England Nuclear. Mira-cloth was from Calbiochem-Behring. Glass double-dis- tilled water was used in all gradients, assays, and homogeni- zation media. IEF Apparatus. The apparatus shown in Fig. 1 was an ad- aptation of the design by Sherbet for IEF-M3 (7). In contrast to the solid-piece construction of Sherbet, two separate tubes of Plexiglas were used to separate the cathode com- partment from the gradient and sample, thereby allowing more efficient cooling of the apparatus. A Teflon blocking valve was used to close off the cathode compartment during fractionation of the gradient. Electrofocusing. The blocking valve was moved to the open position and 2.2 ml of cathode solution [30% (wt/wt) sucrose/6% (wt/vol) NaOHI/2% (vol/vol) ethanolamine for broad-range pH experiments; 30% sucrose/1.6% (vol/vol) 7/9 ampholyte for narrow-range pH experiments] was added to the bottom of the larger chamber. A 15-30% (wt/wt) su- crose gradient containing 1% (vol/vol) ampholytes was then layered on top of the cathode solution by using a linear gradi- ent maker. For narrow-range pH columns the low-density sucrose solution for gradient-making contained 0.25 ml of 40% 4/6 ampholyte in 20 ml of 13% (wt/wt) sucrose and the high-density sucrose solution contained 0.22 ml of 40% 4/6 ampholyte and 0.54 ml of 20% 3/5 ampholyte in 20 ml of 30% (wt/wt) sucrose. For broad-range pH columns, the low-den- sity sucrose solution contained 0.25 ml of 40% 3/10 ampho- lyte in 20 ml of 13% (wt/wt) sucrose and the high-density sucrose solution contained 0.75 ml of 40% 3/10 ampholyte in 20 ml of 30% (wt/wt) sucrose. During gradient-making the Abbreviations: IEF, isoelectric focusing; GS I, glucan synthetase Ir GS II, glucan synthetase II; Met-embryos, embryos grown in sul- fate-deficient sea water containing 10 mM methionine; S04-em- bryos, embryos grown in artificial sea water containing 0.1 mM sul- fate. *Present address: Department of Biology, University of Saskatche- wan, Saskatoon, Saskatchewan, Canada S7N OWO. tTo whom reprint requests should be addressed. 4804 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Proc. Natl. Acad. Sci. USAVol. 81, pp. 4804-4808, August 1984Cell Biology

Isoelectric focusing of plant cell membranes(pI determinations/Fucus/Golgi/plasma membrane/polar transport)

LAWRENCE R. GRIFFING* AND RALPH S. QUATRANOtDepartment of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331

Communicated by Winslow R. Briggs, February 29, 1984

ABSTRACT An isoelectric focusing method is describedthat discriminates plant cell organelle populations on the basisof surface charge. The isoelectric points (pI values) of the Gol-gi apparatus, the mitochondria, and putative plasma mem-brane from etiolated pea stem cells are reported. The pI of thepea Golgi apparatus is similar to that of the 35SO4-labeledmembranes of developing Fucus embryos. The pI of Fucus35SO4-labeled membranes depends on whether the membranesare being polarly transported to the growing tip or randomlytransported to the entire periphery of the zygote. Those sub-ject to polar tip transport have a significantly greater negativesurface charge than those being randomly transported. Theimplication of this result toward an understanding of the local-ization mechanism is discussed. The method is also capable ofsubfractionating glucan synthetase H-containing membranes(putative plasma membrane) from peas. The pI of putativeplasma membrane from a pea stem homogenate is similar tothe pI of the plasma membrane of whole protoplasts of Cathar-anthus leaf cells. Isoelectric focusing appears to be a usefultechnique to discriminate membranes and, hence, to providenew information and approaches to study cellular and devel-opmental phenomena.

Isoelectric focusing (IEF) is used extensively to purify pro-teins, usually employing a polyacrylamide gel as a supportmedium (1). IEF can also be used to purify substances ofvery high molecular weight if a density gradient is used as ananticonvection medium. The advantages of a density gradi-ent for high molecular weight samples are rapid recovery ofsamples and short focusing time. Several IEF columns oflow volume have been designed for a variety of purposes (2-6). The low-volume density gradient column used in thisstudy is similar to that described by Sherbet (7) for studieson animal cell surface charge. The operational characteris-tics are similar to the IEF-M3 column he described (7).The pI of most plant and animal organelles and membrane

fractions is not known. The phase partition method of pI de-termination has been used to study the surface charge on themembranes of the chloroplast (8). A combination of phase-partition cross-point determination and IEF in a sucrose gra-dient has been used to establish the pI of animal cell mito-chondria (9).

Variation in membrane composition make it likely that thesurface differences of organelles will be reflected as a differ-ence in their pI values. In our studies on the polarity of zy-gotes of the brown alga Fucus, we attempted to apply IEF asa method to demonstrate a difference in the pI of vesiclefractions subject to polar or nonpolar transport. We reporthere details of this procedure and the results from the Fucusexperiment. In addition, we demonstrate how the techniqueis applied to the determination of the pI of organelles andmembrane fractions as well as its potential use for prepara-tive separation and isolation of subcellular particles andwhole plant protoplasts.

MATERIALS AND METHODSPlants and Chemicals. Pea seeds (Pisum sativum L. var.

Alaska) were from Burpee Seed (Riverside, CA). Pea seedswere sown in vermiculite and watered every other day withtap water. They were grown in the dark at room temperatureand all manipulations were done under dim red light. After 8days, the epicotyls were harvested and an 8-mm segmentwas cut from 3 mm below the apical hook after they had beenabraided with emery (aluminum oxide 400, Edmund Scien-tific, Barrington, NJ). Fucus distichus L. receptacles werecollected from Yaquina Head (Newport, OR), and treated asdescribed for the release and synchronous development ofzygotes (10). Catharanthus roseus plants were made avail-able by F. Constabel (Plant Biotechnology Institute, Nation-al Research Council, Saskatoon, Sk., Canada). Cytochromec (type III), dithioerythritol, UDPglucose and cellobiosewere from Sigma. Biolyte carrier ampholytes 3/10, 3/5, 4/6,and 7/9 were from Bio-Rad. [1-3H]Glucose (specific activity,4.5 Ci/mmol; 1 Ci = 37 GBq) and [14C]UDPglucose (specificactivity, 275 mCi/mmol) were from ICN; H235504 (specificactivity, 150 mCi/mmol) was from New England Nuclear.Mira-cloth was from Calbiochem-Behring. Glass double-dis-tilled water was used in all gradients, assays, and homogeni-zation media.IEF Apparatus. The apparatus shown in Fig. 1 was an ad-

aptation of the design by Sherbet for IEF-M3 (7). In contrastto the solid-piece construction of Sherbet, two separatetubes of Plexiglas were used to separate the cathode com-partment from the gradient and sample, thereby allowingmore efficient cooling of the apparatus. A Teflon blockingvalve was used to close off the cathode compartment duringfractionation of the gradient.

Electrofocusing. The blocking valve was moved to theopen position and 2.2 ml of cathode solution [30% (wt/wt)sucrose/6% (wt/vol) NaOHI/2% (vol/vol) ethanolamine forbroad-range pH experiments; 30% sucrose/1.6% (vol/vol)7/9 ampholyte for narrow-range pH experiments] was addedto the bottom of the larger chamber. A 15-30% (wt/wt) su-crose gradient containing 1% (vol/vol) ampholytes was thenlayered on top of the cathode solution by using a linear gradi-ent maker. For narrow-range pH columns the low-densitysucrose solution for gradient-making contained 0.25 ml of40% 4/6 ampholyte in 20 ml of 13% (wt/wt) sucrose and thehigh-density sucrose solution contained 0.22 ml of 40% 4/6ampholyte and 0.54 ml of 20% 3/5 ampholyte in 20 ml of30%(wt/wt) sucrose. For broad-range pH columns, the low-den-sity sucrose solution contained 0.25 ml of 40% 3/10 ampho-lyte in 20 ml of 13% (wt/wt) sucrose and the high-densitysucrose solution contained 0.75 ml of40% 3/10 ampholyte in20 ml of 30% (wt/wt) sucrose. During gradient-making the

Abbreviations: IEF, isoelectric focusing; GS I, glucan synthetase IrGS II, glucan synthetase II; Met-embryos, embryos grown in sul-fate-deficient sea water containing 10 mM methionine; S04-em-bryos, embryos grown in artificial sea water containing 0.1 mM sul-fate.*Present address: Department of Biology, University of Saskatche-wan, Saskatoon, Saskatchewan, Canada S7N OWO.tTo whom reprint requests should be addressed.

4804

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

Proc. NatL. Acad. Sci. USA 81 (1984) 4805

gas outletDE + CATHODE

I banana jackr- S lgas outlet

- ~~~~~~teflIon cap

platinum electrodes

i

acrylic support plate

5/1 inch (outer diameter)-1 ~~acrylic lube

12.7

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I 5/8 inch (outer diameter)acrylic tube

4.8 mm

1/4 inch acrylic plate

1/4 inch teflon rod andblocking valve

inch acrylic plate

~~~~~~~~~~~~~~~~~~~~~~~~~~1-.Fc1lamp end of teflIon rod

_rurbber tube

FIG. 1. Design of the IEF column. The inner diameters of thePlexiglas tubes are those that should be used for the solution vol-umes given in the text. Another column with a large tube inner diam-eter of 16 mm and a small tube inner diameter of 6 mm has been usedin some of the experiments, with the appropriate volume compensa-tions in the anode, cathode, and gradient solutions. No difference inperformance between these two column types was detected. Oneinch = 2.54 cm.

cathode solution was displaced into the cathode compart-ment. The anode solution (0.5% orthophosphoric acid) wasthen layered on top, in the region above the acrylic supportplate. The apparatus was attached to a ring stand supportand transferred to a 4°C cold room, where all subsequentoperations were conducted.The column was pre-equilibrated prior to use by running a

constant current (2 mA) for a minimum of 4 hr. The pH andsucrose gradients were stable if left overnight. The pelletedmembrane sample (see below) was resuspended in 22%(wt/wt) sucrose containing 1% of the ampholyte in the col-umn just prior to loading the sample on the column. The 100-,.l sample was loaded with a microcapillary pipette equippedwith a screw plunger over a period of about 2 min. Loadingcaused no detectable disturbance of the gradients. The sam-ple was then electrophoresed at 450 V (1 mA) for 20 minwhen using broad-range pH columns and at 600 V (0.5 mA)for 1.25 hr when using narrow-range columns. The columnsshould never exceed 1 W power dissipation.

Fractionation was done immediately after the membraneshad reached their pI because of possible sedimentation of themembranes as a result of aggregation. After the current wasstopped, the cathode solution blocking valve was closed andfractions were collected dropwise from the bottom of thegradient through the exit port (Fig. 1). The fractions were

then centrifuged for 30 min at 40,000 x g (18,000 rpm in aSorval RC2-B refrigerated centrifuge using an SS34 rotor).The pellets were resuspended in homogenization medium inthe absence [for glucan synthetase II (GS II) assay] or pres-ence of MgCl2. The supernatant was used for pH and densitydetermination at room temperature.

Preparation of Pea Membranes. Etiolated pea segmentswere incubated in [3H]glucose (2.5 ,uCi/ml) in 10 mM Tris/2-(N-morpholino)ethanesulfonic acid, pH 7.0, buffer for 15min and then were homogenized with a razor blade at about5 strokes per s. The homogenization medium contained 40mM Tris/2-(N-morpholino)ethanesulfonic acid, pH 6.5, 1mM dithioerythritol, 1 mM EDTA, 3 mM MgCl2, 10 mMKCl, and 0.25 M sucrose. There were 100 segments used persample in a total volume of 3 ml of homogenization medium.Homogenization, centrifugation, and fractionation were per-formed at 40C. After Mira-cloth filtration, the homogenatewas layered on a 20-60% (wt/wt) linear sucrose gradient andcentrifuged at 27,000 rpm in a Beckman SW28 rotor in aBeckman L8 ultracentrifuge for 3 hr. The gradient contained40 mM Tris, pH 8.0/1 mM dithioerythritol/1 mM EDTA/0.1mM MgCl2. The gradient was fractionated into 75-drop frac-tions (2 ml) by using a Gilson microfractionator and the frac-tions were diluted in a gradient medium without sucrose andcentrifuged at 40,000 x g for 30 min. The fractions were re-suspended in homogenization medium (-MgCl2 for GS II as-say) or the appropriate ampholyte mix. One-third of themembrane pellets from 100 segments was loaded onto theIEF column.

Preparation of Fucus Membranes. Zygotes were incubatedin artificial sea water containing either 0.1 mM sulfate (S04-embryos) or 10 mM methionine (Met-embryos). Both popu-lations were grown in the light at 15°C until each developednormal rhizoids. They were then pulsed (15 min) with 35SO4(2.25 ,uCi/ml) in 0.1 mM S04 artificial sea water. The cellswere homogenized after extensive rinsing in homogenizationmedium [50 mM Pipes-KOH, pH 7.0/10 mM KCl/1 mMEDTA/1 mM dithioerythritol/1 mM MgCl2/0.25 M su-crose/2.5% (wt/vol) dextran 250/2.5% Ficoll 400/0.4%(wt/vol) dextran 40], 18 strokes of a motor-driven Teflonpestle in a glass homogenization vessel for Met-embryos, 8strokes for S04-embryos. The homogenate was layered overa 7-ml 40% (wt/vol) sucrose solution in 10 mM Pipes-KOHpH 7/10 mM KCl/1 mM EDTA/ 1 mM MgCl2/1 mM dithio-erythritol. After centrifuging at 12,000 x g for 10 min (8500rpm in Sorval HB-4 rotor), the top layer was recovered andcentrifuged at 40,000 x g for 45 min. The pellet was recov-ered and resuspended in homogenization medium withoutFicoll and dextran and centrifuged at 10,000 x g for 20 minto sediment poorly resuspended particles. The supernatantwas pelleted at 40,000 x g for 30 min and resuspended insucrose/ampholyte.Enzyme Assays. Cytochrome c oxidase was assayed as de-

scribed (11). Glucan synthetase I (GS I) and GS II were as-sayed also as described (12).Preparation of C. roseus Leaf Protoplasts. The protoplasts

were prepared as in ref. 13. The protoplasts were washedseveral times in 0.4 M mannitol prior to resuspending in su-crose/ampholyte and loading on the column. After loadingabout 104 cells in 100 ul at the pH 7 range, the protoplastsremained on the column for 40 min at 1 W power dissipation,300 V. The OD652 was measured to detect the chlorophyll-containing protoplasts after lysing each fraction with 15 ,ul of10% Triton X-100.

RESULTSIdentification of Pea Organelles. To identify the organelles

studied, three membrane markers were used. GS I is a mark-er enzyme for the Golgi apparatus in plant cells, whereas GS

ANOI

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Cell Biology: Griffing and Quatrano

4806 Cell Biology: Griffing and Quatrano

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FIG. 2. Pea organelle separation by isopycnic centrifugation in a20-60% (wt/wt) sucrose gradient. (A) Representative experiment inwhich pea segments were labeled for 15 min with [3H]glucose priorto homogenization. Membranes in the fractions from the gradientwere pelleted and assayed for incorporation of [3H]glucose (3H cpm,

a), GS II activity ('4C cpm, a), and cytochrome c oxidase activity(AA550/min, A). Background counts of 200 cpm per fraction weresubtracted from the values shown for the GS II results. (B) Repre-sentative experiment in which fractions were directly assayed forGS I ("4C cpm, a). The membrane fractions were then pelleted andassayed for cytochrome c oxidase (AA550/min, A).

II may be associated with the plasma membrane (12). Cyto-chrome c oxidase activity is associated with the mitochon-dria (14). The profiles of these marker enzymes after centrif-ugation of a pea cell homogenate to isopycnic density isshown in Fig. 2. The buoyant density of each membranepopulation is as described (14). By comparing Fig. 2 A withB, these membranes labeled by in vivo incubation of peastem segments with [3H]glucose for short periods (Fig. 2A)have a similar buoyant density to GS I-containing mem-branes (Fig. 2B). Ray et al. (15) have also shown that a shortpulse of [3H]glucose will label only the Golgi apparatus inpea stem segments. As expected, there is a great degree ofoverlap of the GS II-containing membranes with the Golgiand mitochondria markers (Fig. 2A). Using the IEF tech-nique, we demonstrate the separation of these organelles andthe measurement of their different pI values even thoughthey have similar buoyant densities.Broad-Range Electrofocusing of Pea Membranes. The two

isopycnic gradient fractions representing the Golgi-i.e., thepeak of in vivo [3H]glucose incorporation in Fig. 2A-werediluted, pelleted, and resuspended in sucrose containing am-pholyte. The membranes were then applied to the pH 7 re-gion of a pre-electrophoresed IEF column containing 1%3/10 ampholyte (broad-range column). After 20 min of elec-trophoresis, the column was fractionated and radioactivitywas determined (Fig. 3A). The Golgi membranes focused toa pI between 4.5 and 4.2.Two isopycnic gradient fractions containing mitochondria

were diluted, pelleted, and resuspended in sucrose contain-ing ampholyte and applied to a pre-electrophoresed broad-range column in the pH 7 region. After 20 min of electropho-resis, the peak oxidase activity was found between pI 5.0and 4.7 (Fig. 3B). When Golgi and mitochondrial membranesisolated by isopycnic centrifugation were mixed in sucrose

01I*. E

C.E_

0InIn

Fraction

FIG. 3. IEF of pea Golgi and mitochondria using broad-rangeampholytes (pH 3-10). (A) Membranes from the isopycnic gradientthat contain the peak of in vivo [3H]glucose incorporation and there-fore represent the Golgi membranes [31.0o and 32.7% (wt/wt) su-crose] were electrofocused for 20 min. The pH for each 20-drop frac-tion was determined (e) and the pelletable radioactivity was counted(3H cpm, o). The pH values of fractions 14 and 15 were 4.48 and4.24, respectively. (B) Membranes from the isopycnic gradient thatcontained the peak of the cytochrome c oxidase activity and there-fore represented mitochondria [39.9o and 42.7% (wt/wt) sucrose]were electrofocused for 20 min. The pH for each 20-drop fractionwas determined (e). The cytochome c oxidase activity (AA550/min,A) in the membrane pellets from each fraction was assayed. The pHvalues of fractions 11 and 12 were 4.99 and 4.69, respectively. (C)Golgi and mitochondrial membrane suspensions from the isopycnicgradient used in A and B were briefly mixed. After electrofocusing,the 20-drop fractions were assayed for pH (e), pelletable cyto-chrome c oxidase activity (AA550/min, A), and pelletable [3H]glu-cose radioactivity (0). The pH values of fractions 12 and 13 were4.68 and 4.29, respectively.

containing ampholyte and applied to the pH 7 region of abroad-range column, a slight separation of the two mem-brane types was apparent (Fig. 3C). The Golgi membranespeaked at a pI of 4.2, whereas the mitochondrial membranespeaked at 4.7. This separation of the two fractions was moredistinct if a narrow-range ampholyte was used (see Fig. 4C).Narrow-Range Electrofocusing of Pea Membranes. Since

the membranes of the organelles of interest had pI values of4.0-5.0, a mixture of 3/5 and 4/6 ampholytes was used togenerate a pH gradient in this region. When Golgi mem-branes were applied to the pH 5 region of a narrow-rangecolumn, they focused to a pl of 4.13 (Fig. 4A). The GS II-containing membranes that contaminated the Golgi from anisopycnic gradient (see Fig. 2) were not enriched at this pL.GS II-containing membranes were present at pl values bothabove (4.4) and below (4.0) the Golgi pI (Fig. 4A) (see Dis-cussion).When run on a narrow-range column, the mitochondria

had a pI of 4.62 (Fig. 4B). This is very similar to the valueobtained by broad-range IEF (Fig. 3B). Under narrow-rangeconditions, a slightly lower pI was found for all fractions inall experiments.

After gentle mixing of the resuspended Golgi and mito-chondrial membranes, the sample was applied in the pH 5region of the gradient and electrofocused (Fig. 4C). Themembrane separation was very clear when using a narrow-range column, even though the mitochondria seemed to have

Proc. NatL Acad ScL USA 81 (1984)

Proc. Natl. Acad Sci. USA 81 (1984) 4807

0c

0

7-

N

E

T-I

5 N-b-1 -02;5_0.~E

U'4 ~ ~~ 1 ~0.1~

2 4 6 8 10 12 14Fraction

FIG. 4. IEF of pea Golgi and mitochondria using narrow range

ampholytes (pH 5.5-3.5). (A) Golgi-enriched membranes from theisopycnic gradient shown in Fig. 2A, containing =60% of the in vivo[3H]-glucose incorporation and =30% of the GS II activity [29.5%and 32.5% (wt/wt) sucrose], were electrofocused for 1.25 hr. ThepH (o), pelletable 3H radioactivity (o), and GS II activity (o) wereassayed for each 20-drop fraction. Background counts of 200 cpmper fraction were subtracted from the values shown for the GS II

activity in each fraction. Fractions 7, 8, 9, and 10 were pH 4.42,4.30, 4.13, and 4.06, respectively. (B) Mitochondria-enriched mem-branes from the isopycnic gradient shown in Fig. 2A containing thepeak of cytochrome c oxidase activity [41.2% and 44.2% (wt/wt)sucrose] were electrofocused for 1.25 hr. The pH (e) and pelletablecytochrome c oxidase (A) were assayed for each fraction. Fraction 6had a pH of 4.62. (C) Golgi and mitochondrial membrane suspen-sions used in A and B were briefly mixed. After electrofocusing, the20-drop fractions were assayed for pH (e), pelletable cytochrome c

oxidase activity (A), and pelletable [3H]glucose radioactivity (o).The pH values of fractions 7 and 10 were 4.53 and 4.21, respective-ly.

a slightly lower pI than in broad-range experiments. Thecleaner separation with the narrow-range (Fig. 4C) com-pared to broad-range (Fig. 3C) suggests that with an evensmaller pH range, better separation can be achieved. Thelower value found for the mitochondrial pI was also foundfor the Golgi membranes that focused in narrow-range col-umns between pI 4.1 and 4.2. These slightly lower valuesmay be due to the higher voltages or longer times used innarrow-range experiments. The membranes appear more ag-gregated and less diffuse under narrow-range focusing condi-tions.Narrow-Range Electrofocusing of Fucus Membranes.

Growing Fucus zygotes in the presence (SO4-embryos) orabsence (Met-embryos) of sulfate results in altered transportpatterns of 35SO4-labeled Golgi vesicles (16). In S04-em-bryos, polar transport of 35S04-labeled polysaccharides tothe growing tip or rhizoid occurs. There is random 35S04incorporation in the peripheral wall in the Met-embryos (16).As shown in Fig. 5, when these membranes were isolated bycentrifugation and then focused, Met-embryos had 35SO4-la-beled membranes with a lower negative charge (pI = 4.2)than membranes from S04-embryos (pI = 4.0). There wasalso a larger spread of charge in the membrane population

co0

x

E0.0CDU,cm

pH

FIG. 5. IEF of 35S04-labeled membranes from rhizoid-growing,18- to 20-hr (F. distichus embryos. Membranes were obtained bydifferential centrifugation from a homogenate of 0.21 ml of packedcell volume of methionine-grown Fucus zygotes (Met-embryos, o)and 0.35 ml of packed cell volume of artificial sea water-grown Fu-cus zygotes (S04-embryos, *). They were electrofocused in sepa-rate, pre-electrophoresed columns for 2 hr. The 12-drop fractionswere collected and assayed for pH and pelletable radioactivity.

from S04-embryos than Met-embryos. The pl values fromboth were similar to the Golgi apparatus of peas (Fig. 4A),which supports previous results that the Golgi is the site ofpolysaccharide sulfation (17) and the origin of vesicles local-ized in the rhizoid (16, 17).Broad-Range Electrofocusing of Catharanthus Protoplasts.

Whole protoplasts isolated from periwinkle leaves wereloaded on a broad-range column and electrofocused to deter-mine the charge on the outer surface of the plasma mem-brane of the cells. By using the same conditions as for broad-range separation of organelles (Fig. 3), the cells focused to apI between 4.3 and 4.6 (Fig. 6).

DISCUSSIONThe p1 values determined for plant mitochondria (4.7-5.0),using our electrofocusing system in a broad-range pH gradi-ent, are very similar to those reported for animal mitochon-dria (p1 = 4.8-5.2). The animal mitochondria values weredetermined by both phase-partition cross-point determina-tion and density gradient IEF (9). However, the use of amarker enzyme for mitochondria and the demonstration ofits activity after electrofocusing in our study extends thework of Ericson (9), who used a nonspecific turbidity assay.This is important when mixtures of membranes are to be sep-arated by IEF.

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0.2 00 0- 4

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FIG. 6. IEF of C. roseus leaf protoplasts using broad-range am-pholytes (pH 3-10) in a supporting sucrose gradient. Protoplastsfrom young periwinkle leaves were layered on a pre-electrophoresedcolumn and electrofocused for 40 min. They reached their pI-i.e.,they stopped moving-just prior to that time. The 20-drop fractionswere collected and assayed for pH (o) and absorbance at 652 nm (-).

Cell Biology: Griffing and Quatrano

4808 Cell Biology: Griffing and Quatrano

As shown in Fig. 3C and Fig. 4C, mixtures of previouslyisolated organelles can be separated. The broad-range (Fig.3C) values confirm those of the narrow range (Fig. 4C).Even though a different ampholyte system is used, the com-parison of the two separations also demonstrates that thebuoyant density of the supporting medium has little to dowith the final pI determination; the mitochondria are in acompletely different density region of the column in Fig. 3C,as compared to Fig. 4C.The subfractionation of GS II-containing membranes (pu-

tative plasma membrane), shown in Fig. 4A, demonstratesthe capacity of this technique to separate membrane mix-tures not easily separable by conventional techniques suchas density gradients (Fig. 2). Subfractionation of the plasmamembrane might be expected if the cytoplasmic side has adifferent charge than the extracellular side. During homog-enization, the plasma membrane probably forms some vesi-cles with the cytoplasmic side exposed and some with theextracellular side exposed. The vesicles with the cytoplas-mic side exposed would be expected to have a similar chargeto Golgi vesicles, since the outer surface of the Golgi be-comes the cytoplasmic surface of the plasma membrane fol-lowing exocytosis. Hence, the appearance of both Golgi andthe GS II-containing membranes at a pI of about 4 would beexpected. That these membranes can be separated from oth-er organelles on the basis of surface charge is also suggestedby the work of Widell et al. (18). In their experiments there isno clear separation of GS I-containing membranes (i.e., Gol-gi) from GS II-containing membranes, suggesting a similarityin surface charge. However, similar aqueous phase-partitionexperiments have been done by Yoshida et al. (19). By vary-ing the ionic concentration of the aqueous phases, they areable to get separation of membranes with the Golgi marker,inosinediphosphatase, from those containing the putativeplasma membrane marker, pH 6.5-Mg2+-adenosinetriphos-phatase.

Confirmation of the surface charge of the extracellularside of the plasma membrane could be made by determiningthe pI of whole plant protoplasts. Although the pI of peastem protoplasts has not been determined, the pI of periwin-kle (C. roseus) leaf protoplasts is about 4.4 (Fig. 6). This isthe same value as one of the pea GS II-containing membranepeaks (Fig. 4A). Further experiments were done to deter-mine the extracellular charge of a variety of plant protoplastsunder a variety of conditions by using the column shown inFig. 1. By using the conditions of the experiment in Fig. 6,protoplasts from another species and from another tissuetype (soybean suspension culture and periwinkle suspensionculture) have a pI range of 4.3-4.6 (unpublished data).We also use this technique to examine differences be-

tween the 35S04-labeled membranes in Fucus embryos thathave different patterns of localization. The polysaccharidesof the Golgi apparatus of Fucus embryos are radioactivelylabeled with a short pulse of 35 o4 (16, 17). The labeled poly-saccharides are polarly secreted at the rhizoid in 18-hr em-bryos grown in SO4, whereas the labeled polysaccharidesappear randomly secreted in 18- to 20-hr Met-embryos (16).It has been hypothesized by Jaffe (20) and others (21) thattip-localized Golgi vesicles are more negatively charged thannonlocalized randomly secreted Golgi vesicles. Hence, theyare localized by their more negative surface charge in re-sponse to the intracellular electric field of the zygotes (20).Two important conclusions can be made from the data inFig. 5. First, the 35S04-labeled membranes of Fucus zygoteshave a surface charge similar to that of pea Golgi. Further

evidence that the S04-labeled membranes are Golgi-derivedwill be presented elsewhere (unpublished data). Second, theMet-embryos have 35S04-labeled membranes with a surfacecharge that is significantly less negative (higher pl) thanthose from S04-embryos. This is consistent with the hypoth-esis that intracellular electrical fields may localize these ves-icles at the rhizoid site (positive pole) by a self-electropho-retic mechanism (20, 21). A difference in surface charge mayalso be responsible for the binding of these vesicles to cyto-skeletal elements (e.g., microfilaments), which are involvedin the localization phenomena (16).

It is apparent from these results that the IEF technique, asdescribed, can be used to separate plant membranes, organ-elles, and protoplasts on an analytical scale and potentiallyon a preparative scale. Separations by this technique, whichare based on surface charge, may be able to discriminatemembranes not distinguishable by conventional methodsand provide new information on cellular and developmentalphenomena.

We thank Dr. Adrian Cutler and Jim Kirkpatrick (Plant Biotech-nology Institute, National Research Council of Canada, Saskatoon,Sk., Canada) for valuable assistance in the experiment shown in Fig.6. This research was supported by Grant PCM 78-20435 from theNational Science Foundation.

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Proc. NatL Acad Sd USA 81 (1984)