Proc. Natl. Acad. Sci. USAVol. 84, pp. 5720-5724, August 1987Biochemistry

Identification of an acetylation site of Chlamydomonas a-tubulin(posttranslational acetylation/epitope mapping/a-tubulin isoforms/microtubule stability)

MICHEL LEDIZET AND GIANNI PIPERNOThe Rockefeller University, 1230 York Avenue, New York, NY 10021-6399

Communicated by David Luck, May 19, 1987 (receivedfor review April 22, 1987)

ABSTRACT An acetylation site of Chlamydomonasaxonemal a-tubulins was identified near, or within, the bindingsite of 6-11B-1i a monoclonal antibody specific for posttrans-lationally acetylated cr-tubulins. In a first approach, axonemalproteins were hydrolyzed by formic acid, cyanogen bromide, orchymotrypsin and analyzed with immunoblots. The smallestax-tubulin peptide retained on nitrocellulose and containingantibody-binding site(s) was found to span amino acids 37-138(a37-138). A smaller antibody-binding peptide, identified asa25-50, was obtained by complete digestion of a-tubulin withchymotrypsin. This fragment was purified by reversed-phaseHPLC and assayed by its ability to bind 6-11B-1 in solution.Determination of the amino acid sequences of a37-138 anda25-50 showed that residue 40 in axonemal a-tubulin iseN acetyllysine. A sequence very similar to Chlamydomonasa25-50 is found in the majority of a-tubulins analyzed so far.However, the corresponding region is markedly divergent insome a-tubulin isoforms from chicken, Drosophila, and yeast.

Progress in the biochemistry and molecular genetics oftubulins and proteins that interact with the microtubules hasbeen reviewed in the proceedings of a recent conference (1).Since microtubules participate in a wide variety of cellularfunctions, much attention has been devoted to analyzingpossible mechanisms of microtubule differentiation. Forexample, a greater resistance to depolymerizing drugs dis-tinguishes specific microtubule structures as well as subsetsof cytoplasmic microtubules. Such differential stability ofmicrotubules has been correlated, in some cases, with theoccurrence of C-terminal detyrosination (2) and posttransla-tional acetylation (3, 4) of assembled at-tubulin. However, theprocess of microtubule stabilization remains unknown.The presence of modified subunits in microtubules can be

monitored by antibodies specific for acetylated (3-9) ordetyrosinated (10) a-tubulin. As a step toward understandingthe role of a-tubulin acetylation in microtubule stabilization,we set out to determine possible acetylation sites by identi-fying the binding site(s) of a monoclonal antibody, 6-11B-1,specific for posttranslationally acetylated a-tubulins.

MATERIALS AND METHODSPreparation of Chlamydomonas Axonemes. Chlamydomo-

nas 137+ cells were grown and axonemes were isolatedessentially as described (11). Axonemes were immediatelysolubilized in 1% NaDodSO4/15 mM 2-mercaptoethanol.Hybridomas. Hybridomas (5) were grown in HT medium as

described (12). 6-11B-1 IgGs were purified from serum-freeculture medium (HB102, New England Nuclear) by proteinA-Sepharose affinity chromatography and 125I-labeled by thechloramine-T method. After labeling, the 6-11B-1 IgG con-centration was 50 ,g/ml and the specific radioactivity was1.3 x 106 cpm/ul.

Gel Electrophoresis, Transfer to Nitrocellulose, and Anti-Body Binding. These were essentially as described (5), exceptthat 7.5-15% acrylamide Neville (13) gradient gels were used.

Electroelutions. Electroelutions were performed in a UEAelectroeluter (International Biotechnologies, New Haven,CT), using the manufacturer's recommended conditions.

Protein Hydrolyses. Proteins (6 mg/ml) were dialyzed for 2hr against 1000 vol of 20 mM Tris HCl (pH 7.5) and thenadded to 3 vol of 100% formic acid (Sigma), or 3 vol of 100%formic acid containing 20 mg of CNBr per ml (MCB Manu-facturing Chemists, Cincinnati, OH). Incubation times were24 hr at 37°C for hydrolysis by formic acid and 3 hr at 250Cfor CNBr hydrolysis. After hydrolysis, the products weredialyzed against 20 mM Tris-HCl (pH 7.5) for 6 hr andredissolved in 1% NaDodSO4. For the chymotryptic diges-tion, the proteins were dialyzed as described above, andchymotrypsin (45 units/mg; Cappel Worthington, cataloguenumber LS01450) was added from a fresh solution (1 mg/ml)in 20 mM Tris HCl (pH 7.5) to a final concentration of 20,ug/ml for partial and 300 ,ug/ml for complete digestions. Thehydrolysis was considered complete because higher concen-trations of the enzyme did not cause further proteolysis.Incubations were carried out for 1 hr at 37°C. All proteolyticagents apparently retained their specificity when used in thepresence of NaDodSO4: amino acid sequence determinationunambiguously identified sites of hydrolysis at Met-36 ina-tubulin after CNBr cleavage, and after Tyr-24 in a-tubulin,and Tyr-310 and Phe-317 in P-tubulin after chymotrypsinproteolysis.NaDodSO4 Removal and HPLC Analysis. After complete

chymotrypsin digestion, NaDodSO4 was precipitated byaddition of KCI to 0.1 M, followed by a 5-min centrifugationin an Eppendorf centrifuge. This treatment led to a loss of"30% of the protein. The samples were analyzed with aShimadzu HPLC system, using a semipreparative Vydac C18reversed-phase column. Two solutions were used for theelution: solution A was 5% acetonitrile (UV grade, Burdickand Jackson, Muskegon, MI) in 0.1% trifluoroacetic acid(Sigma). Solution B was 70% acetonitrile in 0.1% trifluoro-acetic acid. The elution gradient was (90% A + 10% B) to(70% A + 30% B) in 20 min, (70% A + 30% B) to (50% A +50% B) in 40 min, followed by a 10-min wash in 100% B. Theflow rate was 1.5 ml/min and the eluted proteins weredetected by their absorbance at 220 nm.

Antibody-Binding Competition Assay. Polypeptide solu-tions to be assayed (e.g., aliquots of the HPLC fractions)were dried down under vacuum in a glass tube. The materialwas resuspended into 50 ,l of HT medium, and 1 ng ofradioiodinated 6-11B-1 IgGs was added. After 1 hr, thesolution was transferred into protein-coated wells that hadbeen prepared as follows: axonemes solubilized in 1%NaDodSO4 were diluted to a concentration of 10 ,g/ml withphosphate-buffered saline (PBS; 0.01 M sodium phosphate,

Abbreviations: a37-138, a-tubulin amino acid sequence from posi-tion 37 to 138, etc.; PTH-Xaa, phenylthiohydantoin derivative of thecorresponding amino acid.

5720

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Proc. Natl. Acad. Sci. USA 84 (1987) 5721

0.13 M NaCl, pH 6.8), and fifty microliters of the solution wasleft overnight in Immulon2 Removawells (Dynatech, Alex-andria, VA). The wells were emptied and incubated 1 hr atroom temperature in 0.1% bovine serum albumin in PBSbefore the samples were added. The presence of antigenicmaterial in the sample resulted in a partition of 6-11B-1between the proteins bound to the wells and the solubleantigens. An equilibrium was reached after 2.5 hr. The wellswere then washed six times with 0.1% bovine serum albuminin PBS and the radioactivity bound to the wells was mea-sured. The binding activity of a sample was defined as themass of undigested axonemal polypeptides that would lowerthe radioactivity bound to the wells to the same extent as thesample being tested. This assay was not influenced by thepresence of 0.15% NaDodSO4 or 0.1 M KCI. Control exper-iments established that the specificity of 6-11B-1 for acetyl-ated a-tubulin was preserved in this assay.Amino acid sequences were determined by sequential

Edman degradation using a Gas Phase Sequencer (AppliedBiosystems, Model 470A) and a phenylthiohydantoin (PTH)analyzer (Applied Biosystems, Model 120A).

a b c d e.T.

g h j k

-.

43-

26-

18_12-

6-g5

RESULTS

a-Tubulin Amino Acids 37-203 (a37-203) Binds 6-11B-1. Todetermine the location and number of 6-11B-1 binding sites,we first mapped them on large fragments of Chlamydomonasaxonemal a-tubulins. Since we were unable to enzymaticallyhydrolyze a-tubulin eluted from gels, we used wholeaxonemes dissolved in NaDodSO4 as substrates for thevarious proteolytic agents. The fragments thus obtained were

separated by gel electrophoresis, transferred to nitrocellu-lose, and incubated with the antibody. The apparent molec-ular weight of antigenic polypeptides was calculated fromtheir electrophoretic mobility, using molecular weight mark-ers as a reference. As the amino acid sequence of a-tubulinis known (14), we could predict the molecular weight of thefragments that would be generated by specific hydrolysis ofa-tubulin with CNBr, formic acid, and chymotrypsin. Wethen tentatively identified the antigenic polypeptides bymatching their apparent molecular weights with those ofexpected fragments. The identity of the smallest fragmentswas confirmed by determining their amino acid sequences.

Fig. 1 (lane a) shows a silver-stained electrophoretogram ofaxonemal proteins. The two prominent bands are a- and,B-tubulin. Lane g shows an autoradiogram of the immunoblotof the same material after incubation with 6-11B-1. Theantibody binds to a-tubulin (apparent Mr, 53,000; actual Mr,49,600).

Fig. 1 (lane b) shows an electrophoretogram of formic acidhydrolyzed axonemal proteins; lane h shows the autoradio-gram of the corresponding immunoblot. 6-11B-1 binds tointact a-tubulin and to two partially resolved polypeptides ofapparent Mr 35,000 (FA1) and 34,000 (FA2). As formic acidprimarily hydrolyzes Asp-Pro bonds in proteins (15, 16),these fragments were probably generated by cleavage of thesingle Asp-Pro bond in a-tubulin at position 306-307. Theyrepresent the N-terminal portion of a-tubulins (al-306;expected Mr. 33,500) and carry all the 6-11B-1 binding site(s).The difference of electrophoretic mobility of FA1 and FA2 isunexplained. Immunoblots of the same sample were incu-bated with the monoclonal antibodies 3A5 and B-5-1-2,specific for all a-tubulin isoforms (3, 4). Both antibodies binda unique Mr 18,000 fragment (FA3), which is probablya307-451 (expected Mr, 16,000) (not shown).

Fig. 1 (lane c) shows an electrophoretogram of CNBr-digested axonemal proteins; lane i shows the autoradiogramof the corresponding immunoblot. Four fragments, CNBrl-CNBr4, bind 6-11B-1. Their apparent Mrs are, respectively,33,000, 30,000, 23,000, and 19,000, which tentatively identi-

FIG. 1. Immunoblot analysis of Chlamydomonas axonemes di-gested with formic acid, chymotrypsin, and CNBr. Lanes a-f, silverstained; lanes g-l, autoradiograms of immunoblots of similar gels,after binding of 6-11B-1 and 125I-labeled goat anti-mouse antiserum.The lanes were loaded with 4 ,Ag ofChlamydomonas axonemes (lanesa and g), 4 ,ug of formic acid-digested axonemes (lanes b and h), 15,ug of CNBr-digested axonemes (lanes c and i), and aliquots ofelectroeluted CT1 (lanes d and j), electroeluted CT1 after cleavagewith CNBr (lanes e and k). and electroeluted CT-CNBr2 (lanes f and1). The positions of molecular weight markers (x 10-1) are shown on

the left.

fies them as a2-301, a37-301 (or a2-268), a2-203, anda37-203, which have expected Mrs of 32,800, 29,000 (or29,300), 21,800, and 18,000, respectively. a37-203 (CNBr4) isthe expected product of a complete digestion.

Fig. 2 shows the putative locations within the a-tubulinmolecule of the fragments described in this and the nextsections.

cr-37-138 Binds 6-llB-1; Lys-40 is Totally Acetylated. Par-tial digestion of the axonemal proteins with chymotrypsinprior to CNBr hydrolysis allowed us to isolate smallerantigenic fragments. The smallest 6-11B-1-binding fragmentthat could be reproducibly identified on gels after partialchymotrypsin digestion had an apparent Mr of 16,000 and wasnamed CT1. CT1 and neighboring peptides were electro-eluted from the gel, and aliquots were analyzed by gelelectrophoresis and immunoblotting (lanes d and j). 6-11B-1binds mainly to CT1 and to intact a-tubulin. Further hydrol-ysis of CT1 with CNBr generated a few fragments (lane e).Two of these fragments bound the antibody (lane k). One,CT-CNBrl, had an apparent Mr of 16,000 and was probablyundigested CT1. The other, CT-CNBr2, had an apparent Mrof 11,000.CT-CNBr2 was electroeluted and dialyzed against 0.1%

acetic acid. Its purity was assessed by gel electrophoresis andimmunoblotting (lanes f and l). The 10 N-terminal residues ofCT-CNBr2 were found to be Xaa-Xaa-Asp-Xaa-Thr-Ile-Gly-Gly-Gly-Asp. Although contaminants made it impossible toidentify the first two amino acids, the rest of the sequencematched the sequence 39-46 of Chlamydomonas a-tubulin(Asp-Lys-Thr-Ile-Gly-Gly-Gly-Asp). However, the fourthcycle of Edman degradation did not yield any PTH-Lys butan unknown PTH derivative. We synthesized a sample ofPTH-AcLys in the sequencer, using AcLys (Sigma) as a

precursor, and found that it precisely comigrated with theproduct of the fourth cycle. PTH-AcLys had a retention timeof 12.66 min and eluted between PTH-Glu (retention time,

a

Biochemistry: LeDizet and Piperno

f

5722 Biochemistry: LeDizet and Piperno

Cys Met Asn25 36 50

^ . \ .1 -Z

Phe138

Met203

Met Met Asp268 301 306

- u - - U - P-

mSmS U - -~~~~~~~~~~~~~~~~~~~~

i CMT /CT-CNBr1. CT-CNBr2-CT2

FIG. 2. Proteolytic fragments of Chlamydomonas a-tubulins generated with formic acid (FA), cyanogen bromide (CN Br), or chymotrypsin(CT). See the text for the characterization of the fragments. Since Chlamydomonas axonemes may be formed by two a-tubulin isoforms thatdiffer only in amino acids 308 and 366 (14), all the fragments analyzed are homogeneous in their amino acid sequences.

10.16 min) and PTH-Ala (retention time, 13.16 min). Al-though it is possible that another lysine derivative might haveexactly the same chromatographic behavior as PTH-AcLys,it is very likely that the fourth amino acid of CT-CNBr2 isindeed AcLys.The N terminus of CT-CNBr2 is therefore located imme-

diately after Met-36, indicating a CNBr cleavage. The appar-ent Mr of CT-CNBr2 (Mr, 11,000) places the fragment's Cterminus very far from any methionine, suggesting achymotrypsin cleavage, probably at Phe-135 or Phe-138(expected Mr, 10,900 or 11,200). CT1 can now be identifiedas al-138 (expected Mr, 15,300), as it produced CT-CNBr2(a37-138) after cleavage at Met-36.a25-50 Is the Only Chymotryptic Peptide That Binds

6-11B-1. To analyze more precisely the location of the6-11B-1 binding site(s), we generated shorter peptides, toosmall to be analyzed by immunoblotting, and studied themusing the antibody-binding competition assay described inMaterials and Methods.

Fig. 3 shows the amount of antibody bound to the wellsafter incubation with various amounts of either intactaxonemal proteins or proteins digested to completion withchymotrypsin, before or after NaDodSO4 removal. The threecurves are essentially indistinguishable. Therefore, completedigestion with chymotrypsin does not alter the antibody-binding activity of axonemal a-tubulin. Moreover, no activepeptide(s) is selectively lost during the precipitation ofNaDodSO4.

-

-.0

8..ow

0

c 40

0

I-o.01 20-

04-O0 0.08 0.1 0.5 1 5 10

Mass of axonemal proteins In solution (pg)

FIG. 3. Antibody-binding competition activity of axonemal pro-

teolytic digests. The graph shows the amount of radioiodinated6-11B-1 IgGs bound to the wells in the presence of various masses ofintact axonemal proteins (e), axonemal proteins digested to com-pletion with chymotrypsin (+), and chymotrypsin digest after remov-al of NaDodSO4 by KCI precipitation (o). The amount of radioac-tivity bound to the wells is expressed as a percentage of the amountbound in the absence of antigenic material in the liquid phase.

We then analyzed the chymotrypsin digestion products byC18 reversed-phase HPLC, collecting fractions and testingthem for antibody-binding activity. Fig. 4C shows the activityof the different fractions collected during an analyticalchromatography. Most of the input activity could be recov-ered with elution times between 41 and 42.5 min. The originof the traces of binding activity that have longer elution timeswas not investigated. The active fractions (corresponding toelution times from 41 to 42.5 min) from three preparativechromatographies (elution profile shown in Fig. 4A) werepooled and chromatographed under the same conditions(elution profile shown in Fig. 4B). The portion of the eluatecorresponding to the solid area was collected and the aminoacid sequence of the peptides contained in that fraction wasdetermined. The aliquot analyzed contained 200 pmol of theheptapeptide Leu-Thr-Ala-Ser-Ala-Leu-Phe, which matchesthe ,B-tubulin sequence from amino acids 311-317, and 50pmol of an a-tubulin peptide named CT2. Since f3-tubulin hasno antibody-binding activity, the activity of fraction 41-42.5has to be carried by CT2, which therefore contains a bindingsite for 6-11B-1.The amino acid sequence of CT2 that we obtained was

.0

0

c

DAD-

2.%10-

_ _

C

2L0 Time (min.)10 20 30 40 50 60U

FIG. 4. Chromatography of peptides obtained by complete di-gestion of axonemal proteins with chymotrypsin. HPLC was per-formed on a C18 reversed-phase column. The vertical bar on the leftrepresents 0.02 unit of absorbance. (A) Absorbance at 220 nm of theeluate during preparative chromatography. (B) Absorbance at 220 nmof the eluate when pooled active fractions from the first chromatog-raphy were chromatographed again under the same conditions. (C)Activity of the fractions collected during analytical chromatography.

NH2FA 1 /FA2

Tyr451

COOH

-FA3S - - ' CNBr4

U! - ! * -~~~~~~~~~~~~~~~~~~~I CNBr2CNBr1

Proc. Natl. Acad. Sci. USA 84 (1987)

IL 11 11

I / /

-v-

Proc. Natl. Acad. Sci. USA 84 (1987) 5723

Xaa-Xaa-Glu-His-Gly-Ile-Xaa-Pro-Asp-Gly-Xaa-Met-Pro-Ser-Asp-AcLys-Thr-Ile-Gly-Gly-Xaa-Xaa-Xaa-Ala-Phe-Asn. Amino acids indicated as Xaa could not be unambig-uously identified. The identity and position in the sequenceof the 19 identified amino acids exactly match a25-50. Inparticular, AcLys was again found in position 40 of a-tubulin.Approximately 4 pmol of Asn-50 was released as PTH-Asnfrom CT2 and no additional PTH-amino acids were releasedby subsequent cycles. However, because ofthe small amountof material analyzed, we cannot rule out that the shortterminal peptide was washed off the sequencer cup after therelease of Asn-50 and that the C terminus of CT2 was Phe-52or Phe-53, as expected for a chymotrypsin cleavage.

DISCUSSION

An acetylation site of Chlamydomonas a-tubulin was iden-tified near or within the binding site of the antibody 6-11B-1,specific for posttranslationally acetylated a-tubulin. Theidentity of antigenic fragments of a-tubulin could be deducedby considering their amino acid sequences, apparent molec-ular weights, and the specificity of proteolytic agents. Anal-ysis of a-tubulin fragments by Edman degradation revealedthat Lys-40 is totally acetylated. This is in agreement with theobservation that nearly all the a-tubulin in Chlamydomonasaxonemes is acetylated on the e-amino group of a lysine (17,18). Since the antibody 6-11B-1 binds to both a25-50 anda37-138, its binding site is likely to be located in theC-terminal half of a25-50, within a few amino acids ofAcLys-40. Thus, the antibody-binding site created by acet-ylation of Lys-40 is close to or even contains AcLys-40.Although we may have overlooked minor antibody-binding

sites with a much lower affinity, several arguments suggestthat a-tubulins carry only one binding site for 6-11B-1: (i)Complete chymotrypsin digestion did not reduce the anti-body-binding activity of a-tubulin, nor was any activity loston the HPLC column. Furthermore, cr25-50 has most of theantibody-binding activity present in the eluate. This suggeststhat ca25-50 has all the antibody-binding activity of theoriginal a-tubulin molecule. (ii) The data points describing thebinding of 6-11B-1 to axonemes could be aligned in aScatchard plot (data not shown). This may indicate theexistence of a single binding site. However, this method ofanalysis would not bring out the existence of different sites ifthey have the same affinity. (iii) When complex mixtures ofproteins, such as whole cell lysates, are acetylated in vitro byacetic anhydride, only a-tubulin becomes antigenic (4, 5).Therefore, several specific amino acids besides AcLys arerequired in the binding site to allow recognition by 6-11B-1.a25-50 contains only one lysine and no internal repeats;moreover, the rest of the a-tubulin molecule does not containany amino acid sequences identical to parts of a25-50spanning more than three amino acids. This argues in favorof the uniqueness of the binding site carried by ca25-50.Amino acid sequences of a-tubulins from different orga-

nisms are extensively related to each other. Fig. 5 shows theamino acid sequences of various a-tubulins, aligned with theca25-50 sequence of Chlamydomonas. In most organisms,and in particular in every organism known to contain 6-llB-1-reactive a-tubulins (3-9), a sequence similar to that ofChlamvydomonas cr25-50 is found. Hence, this region may bea site of acetylation even if several amino acids are specifi-cally required in addition to Lys-40 to be recognized by thec-tubulin acetylase.

Interestingly, a testis-specific isoform from chicken, anegg- and embryo-specific isoform from Drosophila, and allknown isoforms of Schizosaccharomyces pombe and Sac-charormyces cerevisiae have very different amino acid se-quences in the cr25-50 region, despite their great overallsimilarity with the Chlamydoinonas a-tubulin. These six

ChlamydomonasConsensus 1Consensus 2PhysarumTrypanosomaChicken brainChicken testisDrosophila a1,a3Drosophila a2Drosophila a4S. Pombe (nda2)1S. Pombe (nda2)2S. Cerev. TUBIS. Cerev. TUB3

CLEHG I QPDG**QMPSDKT I GGGDDAFN**

11 **A svVE

sS T

----- - -- - - I SS ***TF PPSS** S A

** U S

**H V S SL ML

GN

S KS KE

**SLKTKEELTASGSSASFPTEMSEVHKNNSYLNDG**Y NP TRSQMS GG S*IHLEDGLSKPK EEG S*HLEDGLSKPK EEG S

FIG. 5. Comparison of a-tubulin amino acid sequences fromvarious organisms with the region corresponding to amino acids25-50 of the Chlamydomonas sequence. Each amino acid is desig-nated by the standard one-letter code. Positions of identity to theChlamydomonas sequence are left blank. [---] indicates the unknownpart of a sequence. Asterisks indicate spaces added to optimize thealignments of the different sequences. The arrowhead points toLys-40 in Chlamydomonas ca-tubulin. The sequences shown arec-tubulins from Chlainydoinonas (14), mouse isoform Ma4 (19),Physarum polycephalum (20, 32), Trvpanosoma rhodesiense (21),chicken brain (22) and testis (23), Drosophila melanogaster (24), S.pombe (25), and S. cerevisiae (26). The sequence from Stylonychia(33) is identical to that of Chlamydomonas. The "Consensus 1"sequence is common to a-tubulins from human keratinocytes andbrain (27), Chinese hamster ovary cells (28), rat brain (29), pig brain(30), and mouse [isoforms Mal, Ma2, Ma3/7, and Ma6 (19)]. The"Consensus 2" sequence is common to c-tubulins from mouse[isoform Ma4 (19)], Macac a (31), and humans (genomic clone Hc44)(31).

isoforms also have a C-terminal region markedly divergentfrom the sequence Val-Asp-Ser-Val-Glu-Gly-Glu-Gly-Glu-Glu-Glu-Gly-Glu-Glu-Tyr found in most ca-tubulins, and fourof them do not contain a gene-encoded C-terminal tyrosine.Thus, the same divergent isoforms may not be substrates foracetylation and for the removal and addition ofthe C-terminaltyrosine, a second posttranslational modification also relatedto microtubule stability (2). This would be of special interestin the case of the chicken or of Drosophila, where thedivergent isoforms are expressed in a tissue-specific manner.The processes of microtubule stabilization correlated withposttranslational acetylation and detyrosination of a-tubulinwould then depend (at least in part) on the control of theexpression of specific ca-tubulin genes.

We wish to thank Dr. Donna Atherton, who determined the aminoacid sequences at the Protein Sequencing Facility of the RockefellerUniversity, Dr. James Manning and Todd Miller for their adviceconcerning the HPLC, Pamela Meinwald for her help in the devel-opment of the antibody-binding competition assay, and ZentaRamanis who grew the Chlamydomonas cells. M.L. is a Reynoldsfellow in the graduate program of The Rockefeller University. G.P.was supported by Grant GM 28702 from the National Institutes ofHealth.

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Proc. Natl. Acad. Sci. USA 84 (1987)