J. Cell Set. 44, 317-333 (1980) 317Printed in Great Britain © Company of Biologists Limited 1980

THE ORAL APPARATUS OF TETRAHYMENA

V. ORAL APPARATUS POLYPEPTIDES AND THEIRDISTRIBUTION

R. H. GAVINDepartment of Biology, Brooklyn College of The City University of Neto York,Brooklyn, N.Y., U.S.A.

SUMMARY

Two-dimensional electrophoresis was used to resolve approximately 162 polypeptides fromthe isolated oral apparatus of Tetrahymena thermophila. The molecular weight range was between110000 and 15000 Daltons. The polypeptides had apparent isoelectric points between pH 3-3and pH 7-2. Electrophoretic analysis of isolated ciliary axonemes and fractionated oral ap-paratuses made possible the assignment of polypeptides to structures within the oral apparatus.Approximately 24 polypeptides, including a. and /? tubulins, are probable components of thebasal body-basal plate complex. At least 5 of the oral apparatus polypeptides, including a and fltubulin, are components of the oral apparatus ciliary axonemes. Approximately 138 poly-peptides are components of the oral apparatus framework.

INTRODUCTION

There is a continuing need for studies that refine our knowledge of the molecularstructure of the ciliate cortex and its constituent organelles. The oral apparatus ofTetrahymena is a cortical feeding structure composed of ciliated and non-ciliated basalbodies interconnected by a framework of microtubules and filaments (Fig. 1). Theultrastructure (Nilsson & Williams, 1966; Forer, Nilsson & Zeuthen, 1970; Wolfe,1970; Rannestad & Williams, 1971; Buhse, Stamler & Corliss, 1973; Gavin, 1977;Sattler & Staehelin, 1979), morphogenesis (Frankel & Williams, 1973) and genetics(Orias & Pollock, 1975; Kaczanowski, 1976; Frankel, Nelsen & Jenkins, 1977;Jerka-Dziadosz & Frankel, 1979; Frankel & Jenkins, 1979) of this cortical organellecomplex have been extensively investigated. However, there have been relatively fewstudies devoted to the molecular composition of the oral apparatus (Rannestad &Williams, 1971; Gavin, 1974; Vaudaux, 1976).

One report (Gavin, 1974) showed that in a one-dimensional urea-polyacrylamidesystem approximately 20 proteins could be resolved from isolated oral apparatuses.In the present paper we report the use of a technique for the separation of poly-peptides in 2 dimensions (O'Farrell, 1975) to resolve approximately 162 polypeptidesfrom the isolated oral apparatus of Tetrahymena. In addition, by making use of afractionation procedure (Wolfe, 1970) that solubilizes oral apparatus basal bodieswhile leaving intact a framework of microtubules and filaments (Gavin, 1977), wehave shown that about 138 oral apparatus polypeptides are constituents of the frame-work, whereas only 24 polypeptides are basal body fraction components.

3 i 8 R. H. Gavin

urn-

- PC

-df

Fig. i. Diagram of the oral apparatus showing the relationship between oral apparatusbasal bodies (open circles) and the fibrillar framework (solid lines). Each membranelle(m) consists of 3 rows of hexagonally arranged ciliated basal bodies. The undulatingmembrane (ura) consists of 2 rows of basal bodies, of which only the outer row isciliated. Membranellar connectives (me) interconnect the 3 membranelles. The anteriorend of each membranelle is connected to the undulating membrane by cross connectives(cc). The posterior end of each membranelle is connected to the undulating membraneby peripheral connectives (pc). The oral ribs (or) originate at the undulating membraneand join with fibres from the third membranelle to form the deep fibre (df).

MATERIALS AND METHODS

Culture methods

Tetrahymena thermophila (Nanney & McCoy, 1976) was grown at 35 °C in a mediumconsisting of 1-5 % bacropeptone (Difco) and 0-4% yeast extract (Difco).

Oral apparatus isolation

The procedures for isolating oral apparatuses were basically those described by Wolfe (1970).However, we have made several minor modifications which we have found to be important forobtaining consistently highly purified oral apparatuses.

Cells were grown in bactopeptone supplemented with yeast extract since proteose peptone-

Oral apparatus polypeptides 319

grown cells yielded lysates containing flocculent debris which was not removed during thecentrifugations through sucrose.

After the addition of the isolation medium and Triton X-100 (Wolfe, 1970), cell contentswere dispersed, resulting in 'ghosts' which retained cell shape. At this stage the preparationwas vigorously stirred (in an ice bath) by means of a magnetic stirrer until all oral apparatusesbecame detached from the disintegrating pellicle. This step was the most time-consuming,frequently requiring 1-2 h. The suspension was then centrifuged as described by Wolfe (1970).The resulting pellet was vortexed on a vortex mixer and resuspended in isolation medium whichcontained 2#o% Triton X-100. All subsequent centrifugations were carried out with 2-o%Triton X-100 in the isolation medium. Prior to the final centrifugation the suspension wasfiltered through a polycarbonate filter (pore size 8-o /tm, Nuclepore Filter Corp., Pleasanton,Calif.).

Ciliary axoneme isolation

Tetrahymena cilia were isolated according to procedures described by Gibbons (1965). Anadded step in the procedure was filtration of the isolated cilia suspension through a poly-carbonate filter as described for oral apparatus isolation. The filtrate was centrifuged at 10000 gfor 30 min to pellet the cilia. In order to effect demembranation of the ciliary axoneme, the wetcilia pellet was resuspended in the Triton X-100 isolation medium that was used for oralapparatus isolation. The suspension was vigorously vortexed and left in an ice bath for 30 min.Subsequently the suspension was centrifuged at 10000 g for 30 min. The axoneme pellet waswashed once with distilled water and retained for electrophoretic analysis.

Oral apparatus fractionation

Isolated oral apparatuses were resuspended in i-o M KC1 and maintained at 4 °C for 18 h.Subsequently, the suspension was centrifuged at ioooog' for 30 min and the supernatant,which contained solubilized basal bodies (Gavin, 1977), was retained for further analysis. Thepellet, which contained the oral apparatus framework (Gavin, 1977), was washed twice indistilled water and retained for further analysis.

Protein assays

Protein assays were performed as described by Lowry, Rosebrough, Farr & Randall (1951)using serum albumin standards.

Radioactive labelling

Cells were grown in a medium consisting of 1 % bactopeptone and 01 % yeast extract andcontaining 5 /iCi/ml of pHJleucine (42 Ci/mM). When the culture reached stationary phasecells were harvested and oral apparatuses isolated. The radioactive cell lysate was retained.

In order to determine the extent to which cellular proteins could be adsorbed to isolatedoral apparatuses during the isolation procedure, non-radioactive cells were lysed in the radio-active lysate retained from the isolation of labelled oral apparatuses. Prior to use, the radio-active lysate was centrifuged at ioooog for 45 min to remove any remaining labelled oralapparatuses. Oral apparatuses were then isolated from cells that were lysed in the radioactivelysate and subsequently assayed for acid-precipitable radioactivity.

For acid precipitations, an aliquot of the desired sample was mixed with 2 ml of cold 10 %TCA and passed through a Millipore filter. The filter was washed with at least 25 ml of cold5 % TCA, dried and dissolved in Bray's solution (Bray, i960) for scintillation counting.

Electrophoresis

Chemicals. Acrylamide, A^iV'-methylene bis acrylamide, and AT.TV.iV'.iV'-tetramethylene-diamine were obtained from Eastman Kodak. Ultrapure urea was purchased from Schwarz/Mann. Ampholines and Coomassie Brilliant Blue R-250 were obtained from Bio-Rad Labora-tories. Glycine, Tris base and ammonium persulphate were obtained from Sigma Chemical Co.

320 R. H. Gavin

Preparation of samples. A wet pellet of isolated oral apparatuses was mixed with approxi-mately 4-5 vol. of solubilizing solution consisting of 8 M urea, o-oi M Tris pH 8-o, and 2 % pHrange 3-10 ampholines or pH range 5-7 ampholines. This solution solubilized more than 90 %of the oral apparatus protein (Gavin, 1974). The sample was vortexed and left in an ice bath for30 min. Subsequently the sample was returned to room temperature and clarified by centri-fugation. A small residue remaining in the bottom of the centrifuge tube was discarded. Thesolubilized protein was then used directly for isoelectric focusing gels. The KCl-soluble oralapparatus fraction was first dialysed at 4 °C against distilled water to remove salt and then driedin vacuo. The powder was dissolved in solubilizing solution as described above. The KC1-insoluble oral apparatus fraction (framework) was solubilized as described for oral apparatuses.All preparations were used for isoelectric focusing within 30 min after solubilization.

Isoelectric focusing. Isoelectric focusing was carried out in glass tubes 5 mm x 140 mm or5 mm x 80 mm using the procedure described by O'Farrell (1975). The gel mixture was asdescribed by O'Farrell except that it contained 5 % ampholines (either pH range 3-10 orpH range 5-7) and detergent was omitted. Gels were prerun for 1 h at 250 V. The sample(500 fig protein in 150 ji\) was then loaded and subjected to electrophoresis at 250 V for 16—18 hat room temperature (24-26 CC).

znd-dimension electrophoresis. Isoelectric focusing gels were extruded from the tubes andimmediately frozen at — 20 °C. The frozen gel was cut transversely into 3 approximately equalsections. Each section was then cut longitudinally into 2 approximately equal segments. Onelongitudinal segment from each section was used for second-dimension analysis. Second-dimension electrophoresis was as described by O'Farrell (1975). The slab gel (3 mm x 10 mm x13 mm) contained either 5, 10 or 15 % acrylamide and a i-cm 4-75 % acrylamide stacking gel atpH 7-2. A segment from a focusing gel (described above) was cemented to the stacking gel bymeans of 1 % agarose made up in stacking gel buffer. A sample well on either margin of theslab gel contained molecular-weight standards. The slab gels were run at a constant current of15 mA until the tracking dye reached the end of the gel.

Gradient analysis. For analysis of pH gradients, segments from a focusing gel were cut intoos-cm slices and placed in 1 ml of carbon dioxide-free water and stored in vacuo for 2 h afterwhich the pH of each fraction was determined with a pH meter.

Staining and destaining gels. If desired, isoelectric focusing gels (without being cut intosections) were fixed for 2 h in 25 % TCA and stained overnight in 025 % Coomassie BrilliantBlue in 50 % methanol and 10 % acetic acid. Destaining was accomplished with a solution ofethanol, acetic acid, water (3 :1:6). Second dimension slab gels were fixed overnight at 4 °C in asolution containing 25 % isopropyl alcohol and 15 % TCA (Fairbanks, Steck & Wallach, 1971).Staining and destaining were as described above.

Data analysis. Measurements for molecular weight and isoelectric point calculations weremade from fully destained, wet slab gels. The slab gel was placed on top of a thin sheet oftracing paper and illuminated from below. A sharpened pencil point was pushed through thecentre of each spot, thereby marking its position on the tracing paper. The result was atracing that retained the precise location of each spot including the molecular weight standards.The tracing paper record was then transferred to coordinate paper for measurement ofmolecular weights and isoelectric points. No attempt was made to quantitate each spot.

RESULTS

One-dimensional analysts: isoelectric focusing

In all experiments reported here isoelectric focusing was carried out at constantvoltage (250 V) for 16-18 h. That these conditions resulted in equilibrium isoelectricfocusing is supported by the following observations: the electrophoretic pattern in thesecond dimension was the same in gels subjected to 4500 V-h or 8500 V-h (data notshown). This system adequately resolved ciliary axoneme tubulins (Fig. 4, p. 323).

The initial experiments were done with broad range ampholines (pH 3-10). Mostof the polypeptides resolved by these gradients had apparent isoelectric points

Oral apparatus polypeptides

5-7 6-0 6-2 6-6i i i i

321

100 k

— 68 k

i 45 k

»

— 25 k

Fig. 2. Photograph of a Coomassie Blue-stained second-dimension gel containingoral apparatus proteins. The entire gel is visible in the photograph. The abscissashows values of the pH gradient from isoelectric focusing. The ordinate shows thepositions of molecular weight markers.

32Z R. H. Gavin

between pH 4-7. The resolution of polypeptides was improved by using a narrowrange (pH 5-7) gradient to amplify the region containing most of the oral apparatuspolypeptides. Resolution was further enhanced by using a 14-cm gel for isoelectricfocusing. Since the available slab gel apparatus for second-dimension analysis accom-modated only an 8-cm gel, it was necessary to cut the 14-cm gel into sections (usually3) and analyse each section separately. Furthermore, cutting each section longi-tudinally enabled the use of one half for second-dimension analysis and the other forgradient analysis.

3-3 4-4 4-8 5-1 5-3 5-6 5-8 6-1 6-4 6-7 70 7-2

— 100k

• •••* , • •* * _ • • _ —68 k

— 45 k

* —25k

Fig. 3. Composite diagram showing the major components consistently resolved onsecond-dimension gels containing oral apparatus proteins. The diagram was madefrom 3 second-dimension gels, each containing a different segment of the samefirst-dimension gel. Details are given in the text. The abscissa shows values of thepH gradient from isoelectric focusing. The ordinate shows the positions of molecularweight markers.

Second-dimension analysis of oral apparatus polypeptides

Fig. 2 is a photograph of the electrophoretic pattern observed when a segment of a14-cm first-dimension gel was analysed in the second dimension on a 10% acryl-amide—SDS slab gel. The gradient analysis from the longitudinal half of the gel thatwas not analysed in the second dimension is shown at the top of the photograph.Approximately 70 polypeptides were resolved in this segment.

Fig. 3 is a composite diagram of the major second-dimension spots consistentlyobserved from the 3 segments of a single first-dimension gel. Each segment was

Oral apparatus polypeptides

50 5-3 5-6 5-8 6-0 6-2i i i i i i

323

68 k

• f1 V Si

4

— 45 k• ! -/

' I - '

Fig. 4. Photograph of a Coomassie Blue-stained second-dimension gel containingciliary axoneme proteins. A portion of the gel is not visible in the photograph. Theabscissa shows the values of the pH gradient from isoelectric focusing. The ordinateshows the positions of molecular weight markers.

324 R. H. Gavin

analysed independently. The gradient analysis for each segment is shown at the topof the diagram. Several minor polypeptides have been excluded from the diagram.Two polypeptides have been designated a and /? tubulin based on their co-migrationwith ciliary axoneme tubulins (Figs. 4, 5).

Distribution of polypeptides within the oral apparatus: ciliary axonemes

It was desirable to determine which oral apparatus polypeptides are constituents ofspecific oral apparatus organelles, e.g. cilia, basal bodies. During the process of oralapparatus isolation cilia are detached (Wolfe, 1970) leaving only a short segment of

4 6 5-3 5 7 5-9 6 2 6-4 7 0~l I I I I 1 T~

• —100k

— 68 k

— 45 k

— 25 k

Fig. 5. Composite diagram showing the major components consistently resolved onsecond-dimension gels containing ciliary axoneme proteins. The diagram was madefrom 2 second-dimension gels, each containing a different segment of the samefirst-dimension gel. Details are given in the text. The abscissa shows the values of thepH gradient from isoelectric focusing. The ordinate shows the positions of molecularweight markers.

axoneme attached to the basal body (Rannestad & Williams, 1971; Gavin, 1977). Inorder to determine which oral apparatus polypeptides are ciliary axoneme compo-nents, isolated cilia from the entire cell were demembranated by using the samesolutions as for isolating oral apparatuses.

Fig. 4 is a photograph of a second-dimension gel containing ciliary axonemeproteins. The 2 tubulins are clearly resolved. The a subunit (right) had a molecularweight of approximately 57000 Daltons and an apparent isoelectric point of 5-4. TheP subunit (left) had a molecular weight of approximately 56000 Daltons and an

Oral apparatus polypeptides 325

apparent isoelectric point of 5-2. There are other axoneme components but they arenot evident in this photograph because of their light staining intensity.

Fig. 5 is a diagram of the major second-dimension spots consistently resolved fromciliary axoneme proteins. There is an obvious absence of high molecular weightproteins, e.g. dyneins, from these ciliary axoneme preparations. Their absence mightbe attributed to the presence of 1 niM EDTA in the solutions used to demembranatecilia. Dyneins are known to be soluble in Tris-EDTA (Gibbons, 1963). Analysis ofthe data summarized in Figs. 7 and 5 revealed that 5 polypeptides, including a and /?tubulins, are common to axoneme and basal body fraction preparations. The re-maining 10 axoneme components are apparently dissimilar from any oral apparatuscomponent.

Table 1. Extraction of isolated oral apparatuses with 1 -o M KCl

Duration of % of solubilizedextraction, h protein

1 2i-67±i-i6S 32-39 ±i-49

18 51-3012-7024 50-5112-03

Isolated oral apparatuses were resuspended in 1 -o M KCl at 4 °C. After each extraction theamount of solubilized protein was determined by the method of Lowry et al. (1951). Thepercentage of solubilized protein is expressed as the mean and standard error of 3 separateexperiments.

Distribution of polypeptides within the oral apparatus: basal body fraction

To show further the distribution of polypeptides within the oral apparatus we madeuse of a limited fractionation procedure first described by Wolfe (1970). Whenisolated oral apparatuses were extracted with 1 -o M KCl approximately 50 % of thetotal oral apparatus protein was solubilized (Table 1). Electron microscopy of thinsections revealed that KCl-extracted oral apparatuses did not contain basal bodies(Gavin, 1977). What remained after KCl-extraction was a framework of microtubulesand filaments which retained the characteristic shape of the oral apparatus (Gavin,1977). The extent to which KCl-extraction removed structures other than the basalbody-basal plate complex (with attached segment of ciliary axoneme), e.g. the densematerial at the proximal end of each basal body, has not been determined. Forpurposes of discussion the KCl-soluble material will be termed basal body fraction,whereas the KCl-insoluble material will be referred to as the framework fraction.

Fig. 6 is a photograph of the tubulin region of a second-dimension gel containingbasal body fraction proteins. The gradient analysis is shown above the photograph.Two components have been designated a and /? tubulin based on co-migration withciliary axoneme tubulins.

Fig. 7 is a diagram of the major second-dimension spots consistently resolved frombasal body fraction proteins. The data summarized in Figs. 3 and 7 show that each

326 R. H. Gavin

4-i5 5-1

i5-3

i5•71

68 k

Tubulins

— 45 k

— 25 k

Fig. 6. Photograph of a Coomassie Blue-stained second-dimension gel containingbasal-body fraction proteins. Portions of the gel (lower and right margins) are notvisible in the photograph. The 2 tubulins are located to the left of the arrow. Alphatubulin is more intensely stained than /? tubulin. Note the similarity in shape betweenthese tubulin polypeptides and the tubulins resolved from ciliary axonemes in Fig. 4.The abscissa shows the values of the pH gradient from isoelectric focusing. Theordinate shows the positions of molecular-weight markers.

Oral apparatus polypeptides 327

polypeptide resolved from basal body fraction proteins was also resolved from oralapparatus proteins.

Distribution of polypeptides within the oral apparatus: framework fraction

Fig. 8 is a photograph of the tubulin region of a second-dimension gel containingframework proteins. The designation of tubulins is based on co-migration with ciliaryaxoneme tubulins. In Fig. 9 is shown a composite diagram of the major second-dimension spots consistently resolved from framework proteins. The data summarizedin Figs. 3 and 9 show that each polypeptide resolved from the framework fraction wasalso resolved from oral apparatus proteins.

3-3 4-4 4-8 51 5-3 5-6 5 8 6-1 6-4 6-7 7-0 7-2

— 100k

— 68 k

— 45 k

— 25 k

Fig. 7. Composite diagram showing the major components consistently resolved onsecond-dimension gels containing basal-body fraction proteins. The diagram wasmade from 3 second-dimension gels, each containing a different segment of the samefirst-dimension gel. Details are given in the text. The abscissa shows the values of thepH gradient from isoelectric focusing. The ordinate shows the positions of mo-lecular weight markers.

Purity of oral apparatus preparations

The following experiment was done to determine the extent to which isolated oralapparatuses contained adsorbed contaminants. Non-labelled cells were mixed with a[3H]leucine-labelled cell lysate, and oral apparatuses were isolated and analysed forradioactivity. If the oral apparatuses contained significant radioactivity its source mustbe proteins that were adsorbed from the labelled cell lysate. However, lack of signifi-cant radioactivity in the oral apparatuses should indicate lack of significant adsorption

R. H. Gavin

5-0 5-1 5-4 5-5

Tubulins

— 45 k

— 25 k

Fig. 8. Photograph of a Coomassie Blue-atained gel containing framework fractionproteins. Portions of the gel (upper and lower margins) are not visible in the photo-graph. The 2 tubulins are located to the left of the arrow. Alpha tubulin is moreintensely stained than /? tubulin. Note the similarity in shape between these tubulinpolypeptides and the tubulins resolved from ciliary axonemes. The abscissa shows thevalues of the pH gradient from isoelectric focusing. The ordinate shows thepositions of molecular weight markers.

of labelled lysate proteins. In each experiment 25 ml of labelled lysate (43 cpm//*g),which had been derived from approximately 2x10 ' cells, was mixed with approxi-mately io7 non-labelled cells. The results of 3 experiments utilizing the same biologicalpreparation are shown in Table 2. The data show that 1 fig of oral apparatus proteincontained as much as c-00022/jg of adsorbed labelled lysate protein. A typicalelectrophoretic analysis utilized 500 fig of oral apparatus protein which theoreticallycontained as much as o-n fcg of adsorbed contaminants. O'Farrell (1975) reportedthat Coomassie Blue can detect o-oi fig of protein. However, Ames & Nikaido (1976)suggested that its sensitivity is somewhat decreased in a 2-dimensional system and

Oral apparatus polypeptides 329

that the limit is probably o-i fig. Assuming a sensitivity of o-oi fig, the contaminationcontained in 500 fig of oral apparatus protein would account for no more than 12 spotsin the second dimension. If the sensitivity of Coomassie Blue is o-i fig, then thecontaminants contained in 500 fig of protein would be undetected in the seconddimension.

33 44 4-8 5-1 5-3 5-6 5-8 6-1 6-4 6-7 70 7-2

— 100 k

• • —68 k• • • • •

• * • • •

. . * • • 2 • . — 45 k• • • •

• • •—25k

Fig. 9. Composite diagram showing the major components consistently resolved onsecond-dimension gels containing framework fraction proteins. The diagram wasmade from 3 second-dimension gels, each containing a different segment of the samefirst-dimension gel. Details are given in the text. The abscissa shows the values of thepH gradient from isoelectric focusing. The ordinate shows the positions of molecularweight markers.

Table 2. Adsorption of cellular protein to isolated oral apparatuses

bp. act. ofisolated oral Amount ofapparatuses, protein adsorbed,

Exp. no. cpm//tg fig/f^g O.A. protein

1 00094 0000222 00066 0000153 00076 000018

CKL 44

330 R. H. Gavin

DISCUSSION

Two-dimensional resolution of oral apparatus polypeptides

The 2-dimensional electrophoretic system described by O'Farrell (1975) was usedto resolve 162 polypeptides from the isolated oral apparatus of Tetrahymena. Severalminor components were excluded from the analysis. The analysis is still not completebecause the deep fibre bundle (Fig. 1) and most of the ciliary axonemes are detachedfrom oral apparatuses during the isolation procedure. This analysis increases byalmost 10-fold the number of components resolved in a i-dimensional system (Gavin,1974). The resolution of such a large number of polypeptides is not too surprisingin the light of the demonstration that Chlamydomonas flagellar axonemes containmore than 130 polypeptides (Piperno, Huang & Luck, 1977). Furthermore, the oralapparatus is not a single organelle but a system of interconnected organelles.

Although the possibility that proteolysis of isolated oral apparatuses influenced thepolypeptide analysis has not been unequivocally excluded, several observationssuggest that it was not involved: (1) Storing isolated oral apparatuses at 4 °C forvarying periods up to 24 h prior to electrophoretic analysis did not alter the observedsecond-dimension pattern. (2) The extraction time required to solubilize oral apparatusbasal bodies could be extended from 18 h to 24 h without any change in the second-dimension pattern. (3) Framework preparations yielded reproducible second-di-mension maps even though they were subjected to variable periods at 4 °C (up to24 h) before electrophoretic analysis. (4) The second-dimension patterns of basal bodyfractions and framework fractions together are the same as the pattern from the oralapparatus from which they were derived.

Considering the large number of polypeptides resolved from the isolated oralapparatus it was important to rule out contamination from other cellular proteins asa possible source of some of these polypeptides. Such contaminants could be derivedfrom either cytoplasmic structures in isolated oral apparatus preparations or fromsoluble proteins adsorbed to oral apparatuses during isolation. Both possibilities areunlikely. Electron microscopy of thin sections of isolated oral apparatuses did notshow contaminating structures (Gavin, 1977), and an experiment (Table 2) showedthat significant adsorption of cellular proteins did not occur during oral apparatusisolation. Taken together, these data provide evidence that contamination fromnon-oral apparatus proteins did not contribute significantly to the analysis describedin this report.

Several regions of the second-dimension pattern shown in Fig. 3 suggest chargeheterogeneity, i.e. components which separate in the isoelectric focusing dimensionbut which have the same molecular weight in the second dimension. Such chargeheterogeneity could be an intrinsic property of the polypeptides or it could beartifactual.Artifactual charge heterogeneity could be induced by such artifacts as carbamylationdue to cyanate in the urea or deamidation. However, such artifacts were probablyavoided by proper sample preparation as described by O'Farrell (1975). That thesample preparation procedure was not in itself conducive to artifactual charge hetero-geneity is further supported by the observation that ciliary axoneme proteins (Fig. 5)

Oral apparatus polypeptides 331

did not show such patterns of charge heterogeneity, even though they were preparedin the same manner as oral apparatus proteins. Taken together these observationssupport the conclusion that the conditions used in this analysis were not consistentwith the generation of artifactual charge heterogeneity.

The apparent charge heterogeneity is probably an intrinsic property of oralapparatus polypeptides, and it could be the result of phosphorylation or methylationof amino acid residues. Thus, some of the polypeptides resolved are probably post-translational modifications of primary gene products. Post-translational modificationsof oral apparatus polypeptides might be a means of activating polypeptides during oralapparatus assembly. It is possible, too, that charge alterations of oral apparatus poly-peptides might be due to in vivo proteolytic modification during oral apparatusmorphogenesis. The activation of morphogenetic precursor proteins by proteolyticcleavage has been documented in studies on bacteriophage T4 morphogenesis(Laemmli, 1970).

Fractionation of isolated oral apparatuses

The constituent organelles of the oral apparatus (Fig. 1) form 2 distinct patterns:(1) the basal body-arrangement that forms the membranelles and the undulatingmembrane, and (2) the framework of microtubules and filaments that interconnectsthe basal bodies. Fractionation of the isolated oral apparatus with KC1 showed thatthe first pattern could be disrupted while leaving the second pattern intact (Gavin,1977). It is clear that the shape and integrity of the isolated oral apparatus are main-tained by this framework of fibrillar structures.

The 2-dimensional analysis of polypeptides coupled with the fractionation of oralapparatuses has made possible the assignment of oral apparatus polypeptides tostructures within the oral apparatus. Approximately 24 polypeptides, including a and J3tubulin have been resolved from the KCl-soluble basal body fraction. These poly-peptides are the probable components of the basal body-basal plate complex and theshort segment of attached ciliary axoneme. An analysis of ciliary axonemes from theentire cell showed that at least 5 of the components resolved from the basal bodyfraction are probable components of the oral apparatus ciliary axonemes. Theremaining oral apparatus polypeptides (approximately 138), including a and /?tubulin, are framework components. Because the framework contains numerousmicrotubules (Gavin, 1977), we conclude that the tubulins resolved from theframework fraction were derived from framework microtubules.

The identification of oral apparatus tubulins has been made on the basis of co-migration with ciliary axoneme tubulins. Although tubulins from the isolated oralapparatus exhibited a differential response to salt solubility, there is by the criteria ofmolecular weight and isoelectric point no apparent difference between the 2 popu-lations of tubulins. The differences in solubility might be explained by the associationof tubulins with other polypeptides within oral apparatus structures.

Because of its sensitivity and reproducibility, the two-dimensional resolution of oralapparatus polypeptides will be useful for further studies on the ciliate cortex. Forexample it should be feasible to determine the timing of synthesis of oral apparatus

332 R. H. Gavin

polypeptides during the cell cycle and the sequence in which these polypeptidesbecome incorporated into structures during oral apparatus morphogenesis. In addition,studies on evolutionary relationships among ciliates can be done by comparing oralapparatus polypeptides in genetically different strains and mutants of Tetrahymena.

This research was supported by research grant GB 40507 from the National ScienceFoundation and City University of New York PSC-BHE awards 11068 and 11394. Mr HenriLichenstein contributed technical assistance and numerous helpful discussions during the earlyphases of this investigation.

REFERENCES

AMES, G. F. L. & NIKAIDO, K. (1976). Two-dimensional gel electrophoresis of membraneproteins. Biochemistry, N.Y. 15, 616-623.

BRAY, G. A. (i960). A simple efficient liquid scintillator for counting aqueous solutions. Analyt.Biochem. 1, 279-285.

BUHSE, H. E. JR., STAMLER, S. J. & CORLISS, J. O. (1973). An analysis of stomatogenesis by

scanning electron microscopy in Tetrahymena pyriformis strain W during synchronous celldivision. Trans. Am. microsc. Soc. 92, 95-105.

FAIRBANKS, G., STECK, T. & WALLACH, D. (1971). Electrophoretic analysis of the major poly-peptides of the human erythrocyte membrane. Biochemistry, N.Y. 10, 2606—2616.

FORER, A., NILSSON, J. R. & ZEUTHEN, E. (1970). Studies on the oral apparatus of Tetrahymena

pyriformis GL. C. r. Trav. Lab. Carlsberg 38, 67-86.FRANKEL, J. & JENKINS, L. M. (1979). A mutant of Tetrahymena thermophila with a partial

mirror-image duplication of cell surface pattern. II. Nature of genie control. J. Embryol. exp.Morph. 49, 203-227.

FRANKEL, J., NELSEN, E. M. & JENKINS, L. M. (1977). Mutations affecting cell division inTetrahymena pyriformis, syngen 1. II . Phenotypes of single and double homozygotes. DeviBiol. 58, 255-275.

FRANKEL, J. & WILLIAMS, N. E. (1973). Cortical development in Tetrahymena. In The Biology ofTetrahymena (ed. A. M. Elliott), pp. 375-409. London, New York: Dowden, Hutchinsonand Ross.

GAVIN, R. H. (1974). The oral apparatus of Tetrahymena pyriformis, mating type 1, variety 1.I. Solubilization and electrophoretic separation of oral apparatus proteins. Expl Cell Res. 85,212-216.

GAVIN, R. H. (1977). The oral apparatus of Tetrahymena pyriformis, strain WH-6. IV. Obser-vations on the organization of microtubules and filaments in the isolated oral apparatus andthe differential effect of potassium chloride on the stability of oral apparatus microtubules.J. Morph. 151, 239-258.

GIBBONS, I. R. (1963). Studies on the protein components of cilia from Tetrahymena pyriformis.Proc. natn. Acad. Sci. U.S.A. 50, 1002-1010.

GIBBONS, I. R. (1965). Chemical dissection of cilia. Archs Biol., Liige 76, 317-352.JERKA-DZIADOSZ, M. & FRANKEL, J. (1979)- A mutant of Tetrahymena thermophila with a

partial mirror-image duplication of cell surface pattern. I. Analysis of the phenotype.J. Embryol. exp. Morph. 49, 167-202.

KACZANOWSKI, A. (1976). An analysis of the mp gene affected morphogenesis in Tetrahymenapyriformis, syngen 1. J. exp. Zool. 196, 215-230.

LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head ofbacteriophage T4. Nature, Lond. 227, 680-685.

LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L. & RANDALL, R. J. (1951). Protein measure-ments with the Folin phenol reagent. J. biol. Chem. 193, 265-275.

NANNEY, D. W. & MCCOY, J. W. (1976). Characterization of the species of the Tetrahymenapyriformis complex. Trans. Am. microsc. Soc. 95, 664-682.

NILSSON, J. R. & WILLIAMS, N. E. (1966). An electron microscope study of the oral apparatusof Tetrahymena pyriformis. C. r. Trav. Lab. Carlsberg 35, 119-141.

Oral apparatus polypeptides 333

O'FARRELL, P. H. (1975). High resolution two-dimensional electrophoresis of proteins. J. biol.Chem. 250, 4007-4021.

ORIAS, E. & POLLOCK, N. A. (1975). Heat sensitive development of the oral organelle in aTetrahymena mutant. Expl Cell Res. go, 345-356.

PIPERNO, G., HUANG, B. & LUCK, D. J. L. (1977). Two-dimensional analysis of flagellar proteinsfrom wild-type and paralyzed mutants of Chlamydomonas reinhardtii. Proc. natn. Acad. Set.U.S.A. 74, 1600-1604.

RANNESTAD, J. & WILLIAMS, N. E. (1971). The synthesis of microtubule and other proteins ofthe oral apparatus of Tetrakymena pyriformis. J. Cell Biol. 50, 709—720.

SATTLER, C. & STAEHELIN, L. A. (1979). Oral cavity of Tetrahymena pyriformis. A freeze-fracture and high voltage electron microscopy study of the oral ribs, cytosome, and formingfood vacuole. J. Ultrastruct. Res. 66, 132-150.

VAUDAUX, P. (1976). Isolation and identification of specific cortical proteins in Tetrahymenapyriformis strain GL. J. Protozool. 23, 458-464.

WOLFE, J. (1970). Structural analysis of basal bodies of the isolated oral apparatus of Tetra-hymena pyriformis. J. Cell Set. 6, 670—700.

(Received 1 October 1979 - Revised 7 January 1980)