Synthesis Secretion Hydroxyproline Containing ... · Department of Biology, John Muir College,...

Plant Physiol. (1969) 44, 1187-1193

Synthesis and Secretion of Hydroxyproline Containing Macromoleculesin Carrots. I. Kinetic Analysis

Maarten J. ChrispeelsiDepartment of Biology, John Muir College, University of California, San Diego,

La Jolla, California 92037

Received April 9, 1969.

Abstract. When disks of carrot (Daucus carota) phloem parenchyma are incubated for6 days there is a 10-fold increase in cell wall hydroxyproline due to the synthesis andsecretion of hydroxyproline-oontaining macromolecules. The synthesis of these molecules andtheir secretion are demonstrated by measuring the kinetics of incorporation and of chase of14C-proline and hydroxyproline in different fractions of the cytoplasm and the cell wall. Thehydroxyproline-containing molecules which are secreted are associated with the membranousorganelles of the cytoplasm. They oan be fractionated into trichloroacetic acid-soluble andtrichioroacetic acid-precipitated fractions. The properties of the trichloroecetic acid-solublefraotion associated with the membranous organelles are consistent with its role as a cell wallprecursor.

The secretion of carbohvdrates has been studiedin plant cells, especially in relation to cell wallformation (16). However, little is known about pro-tein secretion, undoubtedly because the idea that cellwalls contain proteins has only recently receivedexperimental support [for the most recent review oncell wall proteins, see Lamport (14)]. Proteins orglycoproteins which contain hydroxyproline (hypro)are present in the cytoplasm (19), but occur moreabundantly in the cell wall (5,13). By pulsingtobacco cells in liquid culture with 14C-proline, Olson(18) demonstrated that almost three-fourths of theradioactive cytoplasmic hypro was transferred to thecell wall during chase. In a similar experimentusing Avena coleoptiles, Cleland (3) showed that25 % of the cytoplasmic hypro was transferred. Healso confirmed earlier observations (2, 10) that partof the bound cellular hypro is in a form soluble intrichloroacetic acid. Under the conditions used, thetrichloroacetic acid-soluble hypro of Avena coleop-tiles did not contribute to the observed secretion.We present here results of studies on the incorpora-tion of '"C-prolibe and its conversion to hypro, andthe transfer of these bound radioactive amino acidsto the cell wall in disks of carrot phloem parenchyma.We have identified the trichloroacetic acid-solubleproteins of the membranous organelles of the cell asan important cytoplasmic precursor of cell wall hypro.

1 This investigation was supported by AEC ResearchContract AT (11-1)-34 Project 159 and by a grantfrom the Cancer Research Coordinating Committee ofthe UTniversity of California.

Materials and Methods

Fresh carrots (Daucus carota cv.) were pur-chased in local supermarkets and stored at 5°. Disksapproximately 1 mm thick were cut from a cylinderremoved from the phloem with a No. 3 corkborer.Usually 20 disks were incubated in a 50 ml Erlen-meyer flask containing 10 ml of 5 mrm phosphatebuffer pH 6.0 and 50 ,g/ml of chloramphenicol (15).The flasks were shaken at 300 on a rotary shaker for22 to 24 hr unless otherwise indicated. Chloram-phenicol did not have any effect on the processesstudied, since similar results were obtained withdisks cut under sterile conditions incubated with andwithout chloramphenicol.

Hvdroxyproline in the cytoplasm and the cellwall was quantitated after hydrolysis in 6 N HCl for18 hr at 1100. Cell wall hydrolysates were neutral-ized with sodium hydroxiide and hydroxyproline wasdetermined using the method of Kivirikko andLiesmaa (12). This method did not produce reliableresults with hydrolysates of the cytoplasm becauseof an interfering yellow color. In the cytoplasmicproteins hypro was determined by the method ofJuva and Prockop (11).

Two methods of homogenizing carrot disks wereused. When we were interested in the incorporationof label into the cell wall 20 carrot disks were groundexhaustively in 0.5 ml of 0.5 % detergent (NonidetP40, a nonionic detergent from Shell Chemical Com-pany) in a large mortar in the cold. The walls were

sedi'mfented by spinning at 10OOg for 3 miml andwashed 4 times with 1 mm proline. They were then

1187

Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

PLANT PHIYSIOLOGY

extracted with 9 ml of 0.2 m CaCl2 to obtain thesalt extractable cell wall (SECW) fraction. Theremainder of the cell wall was washed twice withwater, yielding the residual cell wall (RCW) fraction.

To keel) the membranous organelles intact thetissue was homogenized in 0.6 M sucrose containing0.01 M phosphate buffer pH 6.0 (usually 2.5 g with2.5 ml). This was further diluted to 12 ml with0.3 M sucrose containing the same buffer. Cell wallsand starch grains were removed by spinning at1500g for 3 min. The cytoplasm was fractionatedinto a membranous fraction (48,0OOg for 30 min ina Sorvall centrifuge) and a supernatant. The mem-brane fraction was resuspended in 5 % trichloroaceticacid; to the cytoplasmic supernatant we added 1:8volume of 50 % trichloroacetic acid. Proteins wereallowed to precipitate for 1 to 2 hr and then removedby centrifugation at 30,000g for 15 min. The tri-chloroacetic acid-soluble macromolecules in the re-sulting supernatant were collected by pouring theentire trichloroacetic acid supernatant through amembrane filter (B4 from Carl Schleicher andSchuell Company).

Radioactive proline (uniformly labeled with 14C.1.8 mc/mg) was obtained from the New EnglandNuclear Corporation. After the disks had beenincubated with 14C proline, they were rinsed 3 timeswith cold 1 mM proline. To determine the radio-activity in the cytoplasmic proteins and the SECWfraction, aliquots were precipitated with an equalvolume of 15 % trichloroacetic acid in the presenceof 1 mM proline, and collected on membrane filters.An aliquot of the resuspended cell wall was mixedwith 4 volumes of alcohol in the presence of 1 mMproline and collected on a membrane filter(EHWPO2500 from Millipore Corporation). Thefilters were dried and the radioactivity was deter-mined with a liquid scintillation counter. To deter-mine the distribution of 14C in pro and hypro, theradioactive proteins collected on a membrane filterwere subjected to acid hydrolysis (6 N HCl for 18hr at 1100). Insoluble humins and the cell wallresidue were removed by centrifugation. The hy-drolysate was taken to dryness at 800 under vacuumin a "Rotary Evapo-Mix". The residue was resus-pended in 2 drops of water and applied to Whatman3 MM chromatography paper. Proline and hydroxy-proline were separated by descending chromatographyfor 18 hr with isopropanol:formic acid:water 15:2:2(4). The resulting chromatogram was cut into 1inch strips and the radioactivity determined by liquidscintillation counting.

Results

1. Increase in Cell Wall Hypro During Incuba-tion. When carrot disks are incubated for a pro-longed period of time (6 days) there are no signifi-cant changes in the fresh weight and the dry weightof the disks. There is (Fig. 1) a 2 and one-half-foldincrease in the protein-bound hypro of the cytoplasm,

3Dw 200I~

w

1001

0~

I,r

2 4 6

DAYS OF INCUBATIONFIG. 1. Increase in the protein-bound hydroxyproline

of the cytoplasm and cell wall of carrot disks incubatedior 2, 4, or 6 days in buffer. Hydroxyproline was de-termined in the acid hydrolysates of the cell wall andthe cytoplasmic proteins precipitated with 4 volumesof ethanol.

0

co0

I

IU)

0

N

0

x

a.IL

20 40 60

MIN. OF INCUBATION

120

FIG. 2. Incorporation of 14C proline in the cytoplas-mic proteins of carrot disks. Lots of 20 carrot diskswere incubated for 24 hr in -buffer; l AtC of 14C prolinewith 0.25 A±moles of carrier proline was added to eachflask and the tissue allowed to incorporate label for thetimes indicated. The experiment in the "inset" was

done without added carriet.

1188

CELL WALL

CYTOPLASM

KYv/


CHRISPEELS-SECRETION OF HYt)ROXYPROL1NE

and more than a 10-fold increase in the protein-boundhypro content of the wall (from 23 ,xg-231 jig ofhypro/g fresh wt). Under the same conditions thenitrogen content of the cell wall rises 2-fold, whilethe amount of protein-bound proline in the wall in-creases from 75 ,g to 153 Mg1g fresh weight.

2. Labeling of the Cytoplasm and the Cell WallWith 14C Proline and Its Contversion; to 14C Hy-droxyproline. WVhen carrier-free 14C proline is givento disks aged for more than 2 hr the radioactivecompound is taken up very rapidly and incorporatedinto protein. If the incorporation of 14C proline isto be followed for more than 10 min it is necessaryto add an appropriate amount of carrier proline(0.05 ,umoles/hr of incorporation/20 disks). Theincorporation of radioactivity into trichloroaceticacid-precipitated proteins collected on a membranefilter is linear with time and proceeds after a lag ofabout 2 min (Fig. 2) indicating that the amino acidreadily penetrated into the cells of the thin disks andthat the protein-synthesis-precursor-pool of proline

_-A. N_____v__ _ &- .' - 1 - _& 4- - -assumes a

That thewas showinhibitedof cycloheis solubilwhen theto acid hthan 90 °proline or

In plareview sIhydroxylapeptide c

labeled wproteins2 to 3 mi

(A

N

0

X

"I

U)

FIG. 3attached tof the preA is the fsalt (SE((RCW).

Table I. Accumulation of Protein-bound Radioactivityin thc Cytoplasmn antd the Cell Wall

Lots of 20 disks were incubated for varying times,then allowed to incorporate in 1 ,c of 14C proline with0.1 gmole of carrier proline and collected after 30 and90 min. This allowed us to calculate a rate of incor-poration of 14C proline into the cytoplasmic and cellwall proteins.

Radioactivity which accumulatesin fraction

Time of incubation Cytoplasm SECWI RCW2

hr % %1 1112 91.1 7.0 1.9

10 84.9 10.7 4.418 77.5 13.6 8.924 77.0 13.5 9.548 79.0 12.6 8.4

1 Salt extractable cell wall.2 Residual cell wall.

.constant speciic activity in a snort time. We have routinely fractionated the cell wall intoradioactivity is incorporated into proteins 2 parts: a salt extractable fraction (SECW) and a

,n in several ways. First, incorporation is residual fraction (RCW). The labeling of the

more than 95 % by the addition of 10 jig/ml SECW and RCW fractions proceeds at a linear,ximide. Second, the majority of the label rate, but with a time lag of 10 to 15 min (Fig. 3).ized by treatment with pronase. Third. Labeling of cell wall proteins is inllibited more thantrichloroacetic acid-precipitate is subjected 90 % by 10 jtg/nl of cycloheximide and acid hy-Lydrolysis to yield free amino acids, more,,ofthe raiatvt is fon n eihe dlrolysis of the cell walls showvs that 90 % of thehydrofxthepradioactivitye radioactivity is in either proline or hydroxyproline.cydroxyproline. These tests indicate that the radioactivity which is

nt cells (6 18) as in animal cells [for a found in the cell wall is resent in proteins or

ee Udenfriend (21)] proline is probably glycoproteins.

Lted after it becomes incorporated into poly- Exprins:hains.nthe ctoplasm f carro disks xperiments like the ones shown in Fig. 2 and 3

InCpro the cytoplasmn of 14Carrotodi can be used to determine the rate at which protein-

iths behipr the appeaporanceiof of hpro bound label accumulates in the cytoplasm and in thecell wall, and to calculate the percentage of newly

in (inset, Fig. 2). __synthesized protein which is secreted into the wall.

The data in table I show that 90 min after cuttingA B the disks, 7.0 % of the radioactivity appears in the

SECW and only 1.9 % in the RCW. After 24 hrof incubation 13.5 % appears in the SECW and9.5 % in the RCW.

PRO a HYPRO PROa HYPRO The ratio of radioactive lhypro to pro of the 2V//t/ / cell wall fractions increases with time of labeling

indicating that the arrival of 14C hypro in the cellwall lags behind the arrival of 14C pro. For the

YPRO /HYPRO salt extractable fraction the lag is only about 1 or

/x / / 2 min (Fig. 3a). Hypro accounts for 80 % of theradioactivity in that fraction. In the residual cellwall fraction arrival of 14C hypro lags 12 to 15 min

-. . . __________,__, (Fig. 3b) behind the arrival of labeled proteins.20 40 60 120 20 40 60 120 Twenty min after the start of labeling only 17 % of

MIN. OF INCUBATiON the radioactivity is in hypro in the residual cell wallfraction. This increases to 70 % by 180 min.

Arrival of 14C pro and 14C hypro covalently 3. Secreionorotees -bound Hy drxyminto~ ~~ ~ ~~~~~~,marmleueintecl.al.Tispr 3. S-ecretioit of Protein-bound Hydroxyproline.to macromolecules in the cell wall. This is part

!vious experiment and the same conditions apply. Pulse-chase experiments were used to follow theraction of the cell wall which is extractable with secretion of 14C labeled protei.n-bound hypro. It isCW) and B is the residual cell wall fraction first necessary to determine the time required for the

chase to become effective after the carrot disks have

1189


PLANT PHYSIOLOGV'

0C0.CL

co0

ur

a.

x

0~

40O

20O

20 40MIN. OF INCUBATION

60

FIG. 4. Incorporation of 14C proline into carrot disksand chase of the radioactivity from the cytoplasm. Lotsof 2 and one-half g of tissue are incubated for 1 day,then allowed to incorporate 1 ,uc of 14C proline with0.05 Amoles of carrier proline. After 20 min the labelis removed and 100 umoles of carrier proline is added.Tissue is incubated further and samples are collectedat times indicated.

been flooded with non-radioactive proline. Fig. 4shows that there is continued incorporation for 2 to3 min after the chase is applied, assuming there isno dramatic change in the rate of protein synthesis.After that time the total amount of protein-boundradioactivity (cytoplasm plus cell wall fractions)

(I)n

U)

C)

0

N

It

0

x

C-)

6

41

21

20 50 80 140

MIN. OF INCUBATIONFIG. 5. Arrival of 14C proline and 14C hydroxypro-

line covalently attached to macromolecules in the cellwall. Lots of 20 disks are incubated for a day, thenallowed to incorporate 1 Ac of 14C proline in the presenceof 0.05 *moles of carrier proline for 20 min; the labelis removeed and 100 ,Lmoles of carrier proline. is addedas a chase. The tissue is collected at the times indicatedand honiogenized in 0.5 % Nonidet.

remains constanlIt for at least 2 hr. It should bepointed out that hydroxylation probably continuesfor at least 5 nini after chase since hydroxylationlags behind incorporation by 2 to 3 min (Fig. 2).Incorporationi inito the cytoplasmic proteins also coIn-tiniues for 2 to 3 nin, but this is followed byr therapid disappearance of a minor comiiponent whiclhconstitutes 20 to 25 % of the cytoplasmic label atthat time. Althoughl more than four-fifths of thelabel is in lproline. loss of label from hypro accoIlnitsfor half the total loss.

lThe disappearance of labeled l)roteins fronm thecytopllasi1 is matclhedl by the appearance of such)roteins in the cell wall (Fig. 5). Radioactivitvcontinues to arrive in the cell wall long after thechase has become effective: ul) to 30 min afterchase in the SECW fractioni anid up to 60 mimi afterchase in the RCWV fractionl. In the SECW fractionradioactivity arrives at the same rate as in theabsence of a chase (compare with (lashed line) butin the RCW fraction the rate of arrival of radio-activity is greatly reduiced by the chase. However,the total anmount of radioactivity whiclh accumulatesin both fractionis after chase is the equivalent of 15min of secretion at the normal rate. The arrival ofhIyNpro in the cell wall closely parallels the arrival oftotal radioactivitv. In both fractions 80 to 90 % ofthe radioactivity which arrives after chase is inhypro (Fig. 5).

4. Secretion, or T7trnover? We considered thepossibility that the disappearanmce of radioactive hvprofromii the cvtoplasnmic proteins does not constitutesecretion, but rather the miietabolic turniover of theseproteins. It is wvell known that miost plant tissuesdo not containi detectable amounts of free hypro.This is usually cited as evidence that the hypro con-taininig proteins are stable and that the disappearanceof radioactivity in pulse-chase experimenits consti-tutes secretion. WVe must bear in mind the possi-bilitv that hydroxyproline itself could be broken downirapidly and that the hypro containiing macromole-cules need not be broken down to individual aminoacids, but rather to peptides or glvcopeptides. Weattempted to find breakdown products of the hyproconitaininig macromolecuiles wvith these problems inmind.

Carrot disks were allowed to incorporate proline(1 ,c/g of tissue in 10 ml of 2 mM carrier proline)for 20 min, and then chased for 30 and 60 min with10 mM proline. The tissue was homogenized andthe cell walls removed. An aliquot of the cytoplasmwas precipitated with trichloroacetic acid and theproteins collected on a nmembranie filter. The radio-activity in hypro was determined after acid hydrolvsisand chromatography. Another aliquot of the cyto-plasnm (containing both macromiiolecules and smallmolecules) was stubjected to acid hydrolysis and thetotal amiount of radioactive hypro was determined bythe metlhod of Juva and( Prockop (11 ). In thisinethod hvpro is converted to pyrrol wbich is then

TOTAL HYPRO a PRO

CYTOPLASM HYPROa PRO

//

II-L

,/ .' CTOPLASM HYPRO

I*,/

I

I -" SECW' NO CHASE

/_RCW aNO CHASE,'RC SECW(PRaHYR0/, SECW (HYPRO)

1 RCW (PRO a HYPRO)

RCW (HYPRO)

K-1 I .

1190


CHRISPEELS-SECRETION OF HYDROXYPROLINE

Table II. Disappearance of Radioactive HydroxyprolineFront the Cytoplasm During Chase

Carrot disks were labeled for 20 min and then chasedfor 30 to 60 min. "Macromolecular hypro" was obtainedby acid hydrolysis of trichloroacetic acid precipitated pro-teins collected on membrane filters. "Total hypro" wasobtained by acid hydrolysis of the entire cytoplasm.

CPM in HydroxyprolineTime after chase Macromolecules only Total cytoplasm

win0 11,360 11,400

30 4,000 3,76060 3,600 2,940

specifically extracted from the reaction medium.Radioactivity lends increased sensitivity to thismethod an.d it becomes possible to detect 100 cpmof hypro amidst 500,000 cpm of pro.

With both methods we found the same amount ofhypro (table II), indicating that the cytoplasm doesnot contain anv hypro apart from the macromoleculeswhich have been aged for a day contain 2.2 ,ug ofresults were obtained -when the chase was carried outin the presence of 5 mM free hypro.

5. ParticiPation. of a Trichloroacetic Acid-soliubleFractioni. inx the Secrction of Hypro. Carrot diskswhich have been aged for 1 day contain 2.2 jug ofhypro/g fresh weight in the proteins precipitatedwith trichloroacetic acid and 2.0 ug of hypro/g freshweiglht in the trichloroacetic acid-soluble fraction.This *trichloroacetic acid-soluble fractioin is retainedby a bacterial membrane filter indicating that itconsists of rather large molecules. When carrotdisks are labeled with 14C proline approximately10 % of the radioactivity is found in this macro-molecular trichloroacetic acid supernatant fraction.The distribution of radioactivity in pro anid hyprodiffers sharply for the trichloroacetic acid-precipitatedand trichloroacetic acid-soluble fractions. In thetrichloroacetic acid-precipitated proteins about 10 %of the radioactivity is in hypro and 90 % in pro.In the trichloroacetic acid-soluble fraction on theother hand, 65 % of the radioactivity is in hypro and35 % in pro. The trichloroacetic acid-soluble frac-tion therefore accounts for almost half of the radio-active hypro. In a pulse-chase experiment labeledhypro disappears at the same rate from both thetrichloroacetic acid-precipitated proteins and thetrichloroacetic acid-soluble fraction and the cturveswhiclh are obtained are similar to the one presentedin Fig. 3.

Membranous organelles have been shown to beinvolved in the secretion of proteins in several sys-tems (8, 9, 17). Because it is impossible to obtaineven moderately pure particulate fractions from planttissue homogenates by differential centrifugation ofthe cytoplasm, we simply separated the cytoplasm ina "membrane" fraction and a "supernatant" fractionby centrifuginig the tissue homogenate at 48,000gfor 30 min. Both these cytoplasmic fractions contain

trichloroacetic acid-soluble and trichloroacetic acid-precipitable hypro. The trichloroacetic acid-solublehypro of the membrane portion differs in severalways from that of the supernatant. The trichloro-acetic acid-soluble hypro from the membrane fractionrepresents less than 3 % of the total trichloroaceticacid-soluble hypro of the cytoplasm (0.08 ug ofhypro per gram fresh wt). When carrot disks are

labeled for 20 min with 14C proline the distributionsof radioactivity in hypro and pro in the 2 fractionsare completely different. In the membrane trichloro-acetic acid-soluble fraction 85 % to 90 % of theradioactivity is in hvpro, while in the supernatanttrichloroacetic acid-soluble fraction only 15 % is inhypro. Labeling kinetics of the 2 fractions are alsoentirely different. Under conditions where 14C prois continuously incorporated into protein for a periodof 2 hr, incorporation into the membrane trichloro-acetic acid-soluble levels off after 40 min, whilelabeling of the supernatant trichloroacetic acid-solublecontinues for the entire period (Fig. 6). Labelingof the p)roteins precipitated with trichloroacetic acidalso continues for the entire 2-hr period. This sug-gests that the membrane trichloroacetic acid-solublehas a high turnover.

When carrot disks are labeled for 20 min andsubsequently chased. radioactive hypro disappearsrapidlv from the membrane trichloroacetic acid-soluble, but not from the supernatant trichloroacetic

0

a.

co0

a.

x:0

a.

U

UPERNATANT-, TCA SOLUBLE

MEMBRANE-TCA SOLUBLE

20 40 60 120

MIN. OF INCUBATION

FIG. 6. Kinetics of incorporation of 14C prolineinto the membrane trichloroacetic acid-soluble and thesupernatant trichloroacetic acid-soluble fractions of thecytoplasm. Lots of 2 and one-half g of carrots areincubated for a day, then labeled with 1 yC of 14C pro-line and 0.2 ,,moles of proline. The disks are harvestedat the times indicated and homogenized in 0.6 M sucrosewith 0.01 M phosphate pH 6.0. The cytoplasm is cen-trifuged at 48,000g for 30 min to spin down the mem-brane fraction. Addition of trichloroacetic acid andsubsequent centrifugation yields a membrane trichloro-acetic acid-soluble and a supernatant trichloroacetic-acid-soluble.

1191


12LANT PH YSIOLOGY

3

0

0.

:

if)

N

X.0

2

20 40 60

MIN. OF INCUBATION

FIG. 7. Disappearance of 14C hypro from the trichlo-roacetic acid-soluble membrane, trichloroacetic acid-pre-cipitated membrane, and supernatant fractions. Lots of2 and one-half g of tissue are incubated for a day, then-allowed to incorporate 1 Auc of 14C proline in the presenceof 0.05 umole of carrier proline. After 20 min thelabel is removed and 100 umoles of carrier prolineis added as chase. The tissue is collected at the timesindicated and homogenized in sucrose-phosphate. Themembrane fraction is collected by centrifugation at48,000g for 30 min and resuspended in trichloroaceticacid. Trichloroacetic acid is added to the supernatantand the proteins in the various fractions are collectedon membrane filters.

acid-soluble (Fig. 7). It also disappears rapidlyfrom the membrane trichloroacetic acid-precipitate,but not from the supernatant proteins. (In Fig. 7the trichloroacetic acid-soluble and trichloroaceticacid-precipitate of the supernatant are combined.)The non-membrane cytoplasm does not seem to con-

tribute to the secretion of hypro-containing proteins.

Discussion

Whenever experiments of the kind reported inthis paper are performed it is necessary to considerthe pro,blem of cross-contamination between thefractions. Cross-contamination of carrot cell wallswith carrot cytoplasm is readily visible with theunaided eye because contaminated walls are orange.Most methods of grinding the tissue result in con-

taminated walls. The contamination cannot be re-

moved by washing the walls with water, detergentsor salt. On the other hand, grinding small amountsof tissue with detergent or water in a mortar usuallygives white cell walls. When non-radioactive tisuewas homogenized in the presence of radioactive carrotcytoplasm containing 0.5 % Nonidet or when non-

radioactive cell walls (extracted -with salt or not)were mixed with labeled carrot cytoplasm, very little

radioactivity remained bound to the cell walls afterisolation and purification in the usual manner. Thus,cytoplasmic proteins do not become readily bound tothe cell wall when they have been solubilized withdetergent.

The completely different kinetics of labeling andof chase observed for the cell wall and cytoplasmicfractions is the strongest evidence for distinct frac-tions. Indeed, during chase, labeled hypro rapidlydisappears from the cytoplasm and accumulates inthe cell wall. Any cross-contamination which existsbetween these 2 fractions must be minimal. Todispel all doubts about the existence of proline andhydroxyproline containing proteins in the cell wall,we examined by autoradiography carrot disks whichwere labeled with 14C proline and subsequentlyplasmolyzed. Our results show the incorporation oflabel in both the cytoplasm and the cell wall (20).

The walls of cells which are proliferating in anunorganized manner (as calluses or in cell cultures)contain more hypro than cell walls of organizedtissues (14). This observation is partially explainedby our finding of an increase in the hypro contentof the cell wall as a function of time of incubationof tissue disks. Liquid cell cultures and calluses areobtained normally by allowing small pieces of tissueto proliferate on a solid nutrient medium containingthe proper hormonal balance. Initially these cellsprobably undergo a wound response not unlike thatobserved in incubating tissue disks. Indeed, theincrease in the hypro content of the cell wall issimilar whether the disks are incubated in a buffbror on a solid nutrient medium. It would appearthen that the hypro content of the cell wall increasesas part of the wound response which excised tissuesundergo.

Radioactive proline is readily incorporated intothe cytoplasmic proteins and converted to hydroxy-proline. There is a lag of 2 to 3 min betweenincorporation and hydroxylation (Fig. 2). At pres-ent there are no data concerning the cytological siteof hydroxylation. The 2 min time lag could repre-sent the time it takes to finish the protein and moveit to the site of hydroxylation, although the possi-bility that nascent protein is hydroxylated cannotyet be excluded. Our preliminary results'(1) indi-cate that the hydroxylation is carried out by anenzyme which has many characteristics in commonwith a hydroxylase [Hutton et al. (7)] isolatedfrom cells which synthesize and secrete collagen.

The labeling experiments suggest that there isa small pool of cytoplasmic proteins which are con-tinuously synthesized and secreted. Labeled proteinsarrive in the cell wall after a time lag of about 15min. In a pulse-chase experiment, labeled proteinscontinue to arrive in the cell wall for a similar periodof time after the chase has become effective. Thedifferent lag-times of arrival in the wall and *thedifferent kinetics of arrival in the wall during chasefor the various fractions which were examined sug-

I MEMBRANE TCA PPT.

I / MEMBRAN/, TCA-SOLuLU

/ / X- xx

/If/ iERNATANT

I,~~~~~~~~~~~~~~~~~~~~~

1192


CHRISPEELS-SECRETION OF HYDROXYPROLINE

gest that each fraction consists of one or more

distinct proteins. The continued arrival of hypro inthe wall during chase is matched by its disappearancefrom the cytoplasm. This confirms similar observa-tions made by Olson (18) with cultured tobaccocells, and by Cleland (3) with Avena coleoptiles.We were unsuccessful in finding breakdown productsof the hypro containing macromolecules indicatingthat their disappearance from the cytoplasm mostprobably does not constitute metabolic turnover, butrather secretion.

Hydroxyproline in the cell can be fractionatedinto trichloroacetic acid-soluble and trichloroaceticacid-precipitated fractions (2, 3, 10). The trichloro-acetic acid-soluble fraction is non-dializable (3) andis retained by bacterial membrane filters suggestingthat it is macromolecular. We fractionated thecytoplasm into a fraction containing menmbranousorganelles and a supernatant fraction, and thenseparated trichloroacetic acid-precipitated hypro fromtrichloroacetic acid-soluble hypro in each fraction.This allowed us to show that the hypro containingmacromolecules which are secreted are associatedexclusively with the membranous organelles, and thatthey are evenly distributed over the trichloroaceticacid-soluble and the trichloroacetic acid-precipitatedproteins. We have no evidence for a chemical dif-ference between trichloroacetic acid-precipitated andtrichloroacetic acid-soluble hypro. However, becausea large measure of purification is achieved by pre-cipitating the bulk of the proteins with trichloroaceticacid we concentrated first on the characterization ofthe trichloroacetic acid-soluble fraction from themembranous organelles. It has a number of proper-ties consistent with its role as a cell wall precursor:it constitutes only a small amount of the cytoplasmichypro; it is labeled to saturation within 40 mminafter the addition of 14C pro to the carrot disks; thislabel disappears rapidly in a pulse-chase experimentand has a half-life of roughly 20 min; 85 to 90 %of the label is in hypro and 10 to 15 % is in pro;although it is soluble in trichloroacetic acid, it isretained by membrane filters indicating that it iseither a large molecule or an aggregate which is notreadily dissociated. The importance of this fractionas a cell wall precursor warrants its further char-acterization.

Acknowledgments

It is a pleasure to thank Mrs. M. Doerschug andMr. E. Walker for their valuable assistance. MaryEricson, Angeline Douvas, Greg Brewer, and DaveWeaver, all first year graduate students in Biology,contributed to these results during their rotation projects.

Literature Cited

1 CHRISPEELS, M. J., M. R. DOERSCHUG, AND D. SAD-AVA. 1969. Secretion of hydroxyproline contain-ing macromolecules in carrot cells. Abstracts, XIIntern. Botan. Congr. p 32.

2. CLARKE, F. M. W. AND G. M. ELLINGER. 1967.Fractionation of plant material. J. Sci. Food Agr.18: 536-40.

3. CLELAND, R. 1968. Distribution and metabolismof protein-bound hydroxyproline in an elongatingtissue, the Avena coleoptile. Plant Physiol. 43:865-70.

4. CLELAND, R. AND A. M. KARLSNES. 1967. A pos-sible role of hydroxyproline containing proteins inthe cessation of cell elongation. Plant Physiol.42: 66971.

5. DOUGALL, D. K. AND K. SHIMBAYASHI. 1960.Factors affecting growth of tobacco callus tissueand its incorporation of tyrosine. Plant Physiol.33: 396-404.

6. HOLLEMAN, J. 1967. Direct incorporation of hy-droxyproline into protein of sycamore cells incu-bated at growth-inhibitory levels of hydroxyproline.Proc. Natl. Acad. Sci. 57: 50-54.

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