The Oral Apparatus of Tetrahymena V

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

THE ORAL APPARATUS OF TETRAHYMENA V. ORAL APPARATUS POLYPEPTIDES AND THEIR DISTRIBUTION

R. H. GAVIN Department 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 from the isolated oral apparatus of Tetrahymena thermophila. The molecular weight range was between 110000 and 15000 Daltons. The polypeptides had apparent isoelectric points between pH 3-3 and pH 7-2. Electrophoretic analysis of isolated ciliary axonemes and fractionated oral apparatuses made possible the assignment of polypeptides to structures within the oral apparatus. Approximately 24 polypeptides, including a. and /? tubulins, are probable components of the basal body-basal plate complex. At least 5 of the oral apparatus polypeptides, including a and fl tubulin, are components of the oral apparatus ciliary axonemes. Approximately 138 polypeptides are components of the oral apparatus framework.

INTRODUCTION There is a continuing need for studies that refine our knowledge of the molecular structure of the ciliate cortex and its constituent organelles. The oral apparatus of Tetrahymena is a cortical feeding structure composed of ciliated and non-ciliated basal bodies interconnected by a framework of microtubules and filaments (Fig. 1). The ultrastructure (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 organelle complex have been extensively investigated. However, there have been relatively few studies 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-polyacrylamide system 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 polypeptides in 2 dimensions (O'Farrell, 1975) to resolve approximately 162 polypeptides from the isolated oral apparatus of Tetrahymena. In addition, by making use of a fractionation procedure (Wolfe, 1970) that solubilizes oral apparatus basal bodies while leaving intact a framework of microtubules and filaments (Gavin, 1977), we have shown that about 138 oral apparatus polypeptides are constituents of the framework, whereas only 24 polypeptides are basal body fraction components. 3i8 R. H. Gavin

urn-

- PC

-df

Fig. i. Diagram of the oral apparatus showing the relationship between oral apparatus basal bodies (open circles) and the fibrillar framework (solid lines). Each membranelle (m) consists of 3 rows of hexagonally arranged ciliated basal bodies. The undulating membrane (ura) consists of 2 rows of basal bodies, of which only the outer row is ciliated. Membranellar connectives (me) interconnect the 3 membranelles. The anterior end of each membranelle is connected to the undulating membrane by cross connectives (cc). The posterior end of each membranelle is connected to the undulating membrane by peripheral connectives (pc). The oral ribs (or) originate at the undulating membrane and 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 medium consisting 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 for obtaining 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 the centrifugations through sucrose. After the addition of the isolation medium and Triton X-100 (Wolfe, 1970), cell contents were dispersed, resulting in 'ghosts' which retained cell shape. At this stage the preparation was vigorously stirred (in an ice bath) by means of a magnetic stirrer until all oral apparatuses became 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 which contained 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 was filtered 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). An added step in the procedure was filtration of the isolated cilia suspension through a polycarbonate filter as described for oral apparatus isolation. The filtrate was centrifuged at 10000 g for 30 min to pellet the cilia. In order to effect demembranation of the ciliary axoneme, the wet cilia pellet was resuspended in the Triton X-100 isolation medium that was used for oral apparatus 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 was washed 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. The pellet, which contained the oral apparatus framework (Gavin, 1977), was washed twice in distilled 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 and containing 5 /iCi/ml of pHJleucine (42 Ci/mM). When the culture reached stationary phase cells 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 isolated oral apparatuses during the isolation procedure, non-radioactive cells were lysed in the radioactive lysate retained from the isolation of labelled oral apparatuses. Prior to use, the radioactive lysate was centrifuged at ioooog for 45 min to remove any remaining labelled oral apparatuses. Oral apparatuses were then isolated from cells that were lysed in the radioactive lysate 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 cold 5 % 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 approximately 4-5 vol. of solubilizing solution consisting of 8 M urea, o-oi M Tris pH 8-o, and 2 % pH range 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 for 30 min. Subsequently the sample was returned to room temperature and clarified by centrifugation. A small residue remaining in the bottom of the centrifuge tube was discarded. The solubilized protein was then used directly for isoelectric focusing gels. The KCl-soluble oral apparatus fraction was first dialysed at 4 °C against distilled water to remove salt and then dried in 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 or 5 mm x 80 mm using the procedure described by O'Farrell (1975). The gel mixture was as described by O'Farrell except that it contained 5 % ampholines (either pH range 3-10 or pH 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 h at room temperature (24-26 CC). znd-dimension electrophoresis. Isoelectric focusing gels were extruded from the tubes and immediately frozen at — 20 °C. The frozen gel was cut transversely into 3 approximately equal sections. Each section was then cut longitudinally into 2 approximately equal segments. One longitudinal 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 x 13 mm) contained either 5, 10 or 15 % acrylamide and a i-cm 4-75 % acrylamide stacking gel at pH 7-2. A segment from a focusing gel (described above) was cemented to the stacking gel by means of 1 % agarose made up in stacking gel buffer. A sample well on either margin of the slab gel contained molecular-weight standards. The slab gels were run at a constant current of 15 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 into os-cm slices and placed in 1 ml of carbon dioxide-free water and stored in vacuo for 2 h after which the pH of each fraction was determined with a pH meter. Staining and destaining gels. If desired, isoelectric focusing gels (without being cut into sections) were fixed for 2 h in 25 % TCA and stained overnight in 025 % Coomassie Brilliant Blue in 50 % methanol and 10 % acetic acid. Destaining was accomplished with a solution of ethanol, acetic acid, water (3 :1:6). Second dimension slab gels were fixed overnight at 4 °C in a solution 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 were made from fully destained, wet slab gels. The slab gel was placed on top of a thin sheet of tracing paper and illuminated from below. A sharpened pencil point was pushed through the centre of each spot, thereby marking its position on the tracing paper. The result was a tracing 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 of molecular 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 constant voltage (250 V) for 16-18 h. That these conditions resulted in equilibrium isoelectric focusing is supported by the following observations: the electrophoretic pattern in the second dimension was the same in gels subjected to 4500 V-h or 8500 V-h (data not shown). This system adequately resolved ciliary axoneme tubulins (Fig. 4, p. 323). The initial experiments were done with broad range ampholines (pH 3-10). Most of the polypeptides resolved by these gradients had apparent isoelectric points Oral apparatus polypeptides 321 5-7 6-0 6-2 6-6 i i i i

100 k

— 68 k

i 45 k

— 25 k

Fig. 2. Photograph of a Coomassie Blue-stained second-dimension gel containing oral apparatus proteins. The entire gel is visible in the photograph. The abscissa shows values of the pH gradient from isoelectric focusing. The ordinate shows the positions of molecular weight markers. 32Z R. H. Gavin between pH 4-7. The resolution of polypeptides was improved by using a narrow range (pH 5-7) gradient to amplify the region containing most of the oral apparatus polypeptides. Resolution was further enhanced by using a 14-cm gel for isoelectric focusing. 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 (usually 3) and analyse each section separately. Furthermore, cutting each section longitudinally enabled the use of one half for second-dimension analysis and the other for gradient 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 on second-dimension gels containing oral apparatus proteins. The diagram was made from 3 second-dimension gels, each containing a different segment of the same first-dimension gel. Details are given in the text. The abscissa shows values of the pH gradient from isoelectric focusing. The ordinate shows the positions of molecular weight markers.

Second-dimension analysis of oral apparatus polypeptides Fig. 2 is a photograph of the electrophoretic pattern observed when a segment of a 14-cm first-dimension gel was analysed in the second dimension on a 10% acrylamide—SDS slab gel. The gradient analysis from the longitudinal half of the gel that was 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 consistently observed from the 3 segments of a single first-dimension gel. Each segment was Oral apparatus polypeptides 323 50 5-3 5-6 5-8 6-0 6-2 i i i i i i

68 k f • 1 V4 Si

— 45 k • ! -/

'I -'

Fig. 4. Photograph of a Coomassie Blue-stained second-dimension gel containing ciliary axoneme proteins. A portion of the gel is not visible in the photograph. The abscissa shows the values of the pH gradient from isoelectric focusing. The ordinate shows the positions of molecular weight markers. 324 R. H. Gavin analysed independently. The gradient analysis for each segment is shown at the top of the diagram. Several minor polypeptides have been excluded from the diagram. Two polypeptides have been designated a and /? tubulin based on their co-migration with 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 of specific oral apparatus organelles, e.g. cilia, basal bodies. During the process of oral apparatus isolation cilia are detached (Wolfe, 1970) leaving only a short segment of

46 5-3 57 5-9 6 2 6-4 70 ~l I I I I 1 T~ • —100k

— 68 k

— 45 k

— 25 k Fig. 5. Composite diagram showing the major components consistently resolved on second-dimension gels containing ciliary axoneme proteins. The diagram was made from 2 second-dimension gels, each containing a different segment of the same first-dimension gel. Details are given in the text. The abscissa shows the values of the pH gradient from isoelectric focusing. The ordinate shows the positions of molecular weight markers. axoneme attached to the basal body (Rannestad & Williams, 1971; Gavin, 1977). In order to determine which oral apparatus polypeptides are ciliary axoneme components, isolated cilia from the entire cell were demembranated by using the same solutions as for isolating oral apparatuses. Fig. 4 is a photograph of a second-dimension gel containing ciliary axoneme proteins. The 2 tubulins are clearly resolved. The a subunit (right) had a molecular weight of approximately 57000 Daltons and an apparent isoelectric point of 5-4. The P 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 are not evident in this photograph because of their light staining intensity. Fig. 5 is a diagram of the major second-dimension spots consistently resolved from ciliary axoneme proteins. There is an obvious absence of high molecular weight proteins, e.g. dyneins, from these ciliary axoneme preparations. Their absence might be attributed to the presence of 1 niM EDTA in the solutions used to demembranate cilia. Dyneins are known to be soluble in Tris-EDTA (Gibbons, 1963). Analysis of the 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 remaining 10 axoneme components are apparently dissimilar from any oral apparatus component.

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

Duration of % of solubilized extraction, h protein 1 2i-67±i-i6 S 32-39 ±i-49 18 51-3012-70 24 50-5112-03 Isolated oral apparatuses were resuspended in 1 -o M KCl at 4 °C. After each extraction the amount of solubilized protein was determined by the method of Lowry et al. (1951). The percentage of solubilized protein is expressed as the mean and standard error of 3 separate experiments.

Distribution of polypeptides within the oral apparatus: basal body fraction To show further the distribution of polypeptides within the oral apparatus we made use of a limited fractionation procedure first described by Wolfe (1970). When isolated oral apparatuses were extracted with 1 -o M KCl approximately 50 % of the total oral apparatus protein was solubilized (Table 1). Electron microscopy of thin sections revealed that KCl-extracted oral apparatuses did not contain basal bodies (Gavin, 1977). What remained after KCl-extraction was a framework of microtubules and filaments which retained the characteristic shape of the oral apparatus (Gavin, 1977). The extent to which KCl-extraction removed structures other than the basal body-basal plate complex (with attached segment of ciliary axoneme), e.g. the dense material at the proximal end of each basal body, has not been determined. For purposes 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 containing basal body fraction proteins. The gradient analysis is shown above the photograph. Two components have been designated a and /? tubulin based on co-migration with ciliary axoneme tubulins. Fig. 7 is a diagram of the major second-dimension spots consistently resolved from basal body fraction proteins. The data summarized in Figs. 3 and 7 show that each 326 R. H. Gavin 4-5 5-1 5-3 5•7 i i i 1

68 k

Tubulins

— 45 k

— 25 k

Fig. 6. Photograph of a Coomassie Blue-stained second-dimension gel containing basal-body fraction proteins. Portions of the gel (lower and right margins) are not visible in the photograph. The 2 tubulins are located to the left of the arrow. Alpha tubulin is more intensely stained than /? tubulin. Note the similarity in shape between these tubulin polypeptides and the tubulins resolved from ciliary axonemes in Fig. 4. The abscissa shows the values of the pH gradient from isoelectric focusing. The ordinate shows the positions of molecular-weight markers. Oral apparatus polypeptides 327 polypeptide resolved from basal body fraction proteins was also resolved from oral apparatus proteins.

Distribution of polypeptides within the oral apparatus: framework fraction Fig. 8 is a photograph of the tubulin region of a second-dimension gel containing framework proteins. The designation of tubulins is based on co-migration with ciliary axoneme tubulins. In Fig. 9 is shown a composite diagram of the major second- dimension spots consistently resolved from framework proteins. The data summarized in Figs. 3 and 9 show that each polypeptide resolved from the framework fraction was also 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 on second-dimension gels containing basal-body fraction proteins. The diagram was made from 3 second-dimension gels, each containing a different segment of the same first-dimension gel. Details are given in the text. The abscissa shows the values of the pH gradient from isoelectric focusing. The ordinate shows the positions of molecular weight markers.

Purity of oral apparatus preparations The following experiment was done to determine the extent to which isolated oral apparatuses contained adsorbed contaminants. Non-labelled cells were mixed with a [3H]leucine-labelled cell lysate, and oral apparatuses were isolated and analysed for radioactivity. If the oral apparatuses contained significant radioactivity its source must be proteins that were adsorbed from the labelled cell lysate. However, lack of significant 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 fraction proteins. Portions of the gel (upper and lower margins) are not visible in the photograph. The 2 tubulins are located to the left of the arrow. Alpha tubulin is more intensely stained than /? tubulin. Note the similarity in shape between these tubulin polypeptides and the tubulins resolved from ciliary axonemes. The abscissa shows the values of the pH gradient from isoelectric focusing. The ordinate shows the positions 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 approximately io7 non-labelled cells. The results of 3 experiments utilizing the same biological preparation are shown in Table 2. The data show that 1 fig of oral apparatus protein contained as much as c-00022/jg of adsorbed labelled lysate protein. A typical electrophoretic analysis utilized 500 fig of oral apparatus protein which theoretically contained as much as o-n fcg of adsorbed contaminants. O'Farrell (1975) reported that 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 contamination contained in 500 fig of oral apparatus protein would account for no more than 12 spots in the second dimension. If the sensitivity of Coomassie Blue is o-i fig, then the contaminants contained in 500 fig of protein would be undetected in the second dimension. 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 on second-dimension gels containing framework fraction proteins. The diagram was made from 3 second-dimension gels, each containing a different segment of the same first-dimension gel. Details are given in the text. The abscissa shows the values of the pH gradient from isoelectric focusing. The ordinate shows the positions of molecular weight markers.

Table 2. Adsorption of cellular protein to isolated oral apparatuses

bp. act. of isolated oral Amount of apparatuses, protein adsorbed, Exp. no. cpm//tg fig/f^g O.A. protein

1 00094 000022 2 00066 000015 3 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 used to resolve 162 polypeptides from the isolated oral apparatus of Tetrahymena. Several minor components were excluded from the analysis. The analysis is still not complete because the deep fibre bundle (Fig. 1) and most of the ciliary axonemes are detached from oral apparatuses during the isolation procedure. This analysis increases by almost 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 surprising in the light of the demonstration that Chlamydomonas flagellaraxoneme s contain more than 130 polypeptides (Piperno, Huang & Luck, 1977). Furthermore, the oral apparatus is not a single organelle but a system of interconnected organelles. Although the possibility that proteolysis of isolated oral apparatuses influenced the polypeptide analysis has not been unequivocally excluded, several observations suggest that it was not involved: (1) Storing isolated oral apparatuses at 4 °C for varying periods up to 24 h prior to electrophoretic analysis did not alter the observed second-dimension pattern. (2) The extraction time required to solubilize oral apparatus basal bodies could be extended from 18 h to 24 h without any change in the second- dimension pattern. (3) Framework preparations yielded reproducible second-dimension maps even though they were subjected to variable periods at 4 °C (up to 24 h) before electrophoretic analysis. (4) The second-dimension patterns of basal body fractions and framework fractions together are the same as the pattern from the oral apparatus from which they were derived. Considering the large number of polypeptides resolved from the isolated oral apparatus it was important to rule out contamination from other cellular proteins as a possible source of some of these polypeptides. Such contaminants could be derived from either cytoplasmic structures in isolated oral apparatus preparations or from soluble proteins adsorbed to oral apparatuses during isolation. Both possibilities are unlikely. Electron microscopy of thin sections of isolated oral apparatuses did not show contaminating structures (Gavin, 1977), and an experiment (Table 2) showed that significant adsorption of cellular proteins did not occur during oral apparatus isolation. Taken together, these data provide evidence that contamination from non-oral apparatus proteins did not contribute significantly to the analysis described in this report. Several regions of the second-dimension pattern shown in Fig. 3 suggest charge heterogeneity, i.e. components which separate in the isoelectric focusing dimension but which have the same molecular weight in the second dimension. Such charge heterogeneity could be an intrinsic property of the polypeptides or it could beartifactual. Artifactual charge heterogeneity could be induced by such artifacts as carbamylation due to cyanate in the urea or deamidation. However, such artifacts were probably avoided by proper sample preparation as described by O'Farrell (1975). That the sample preparation procedure was not in itself conducive to artifactual charge heterogeneity 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 prepared in the same manner as oral apparatus proteins. Taken together these observations support the conclusion that the conditions used in this analysis were not consistent with the generation of artifactual charge heterogeneity. The apparent charge heterogeneity is probably an intrinsic property of oral apparatus polypeptides, and it could be the result of phosphorylation or methylation of amino acid residues. Thus, some of the polypeptides resolved are probably post- translational modifications of primary gene products. Post-translational modifications of oral apparatus polypeptides might be a means of activating polypeptides during oral apparatus assembly. It is possible, too, that charge alterations of oral apparatus polypeptides might be due to in vivo proteolytic modification during oral apparatus morphogenesis. The activation of morphogenetic precursor proteins by proteolytic cleavage 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 undulating membrane, and (2) the framework of microtubules and filaments that interconnects the basal bodies. Fractionation of the isolated oral apparatus with KC1 showed that the 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 maintained by this framework of fibrillar structures. The 2-dimensional analysis of polypeptides coupled with the fractionation of oral apparatuses has made possible the assignment of oral apparatus polypeptides to structures within the oral apparatus. Approximately 24 polypeptides, including a and J3 tubulin have been resolved from the KCl-soluble basal body fraction. These polypeptides are the probable components of the basal body-basal plate complex and the short segment of attached ciliary axoneme. An analysis of ciliary axonemes from the entire cell showed that at least 5 of the components resolved from the basal body fraction are probable components of the oral apparatus ciliary axonemes. The remaining oral apparatus polypeptides (approximately 138), including a and /? tubulin, are framework components. Because the framework contains numerous microtubules (Gavin, 1977), we conclude that the tubulins resolved from the framework 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 oral apparatus exhibited a differential response to salt solubility, there is by the criteria of molecular weight and isoelectric point no apparent difference between the 2 popu- lations of tubulins. The differences in solubility might be explained by the association of tubulins with other polypeptides within oral apparatus structures. Because of its sensitivity and reproducibility, the two-dimensional resolution of oral apparatus polypeptides will be useful for further studies on the ciliate cortex. For example 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 polypeptides become incorporated into structures during oral apparatus morphogenesis. In addition, studies on evolutionary relationships among ciliates can be done by comparing oral apparatus polypeptides in genetically different strains and mutants of Tetrahymena.

This research was supported by research grant GB 40507 from the National Science Foundation and City University of New York PSC-BHE awards 11068 and 11394. Mr Henri Lichenstein contributed technical assistance and numerous helpful discussions during the early phases of this investigation.

REFERENCES AMES, G. F. L. & NIKAIDO, K. (1976). Two-dimensional gel electrophoresis of membrane proteins. 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 cell division. Trans. Am. microsc. Soc. 92, 95-105. FAIRBANKS, G., STECK, T. & WALLACH, D. (1971). Electrophoretic analysis of the major polypeptides 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 in Tetrahymena pyriformis, syngen 1. II. Phenotypes of single and double homozygotes. Devi Biol. 58, 255-275. FRANKEL, J. & WILLIAMS, N. E. (1973). Cortical development in Tetrahymena. In The Biology of Tetrahymena (ed. A. M. Elliott), pp. 375-409. London, New York: Dowden, Hutchinson and 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 and the 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 Tetrahymena pyriformis, syngen 1. J. exp. Zool. 196, 215-230. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, Lond. 227, 680-685. LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L. & RANDALL, R. J. (1951). Protein measurements with the Folin phenol reagent. J. biol. Chem. 193, 265-275. NANNEY, D. W. & MCCOY, J. W. (1976). Characterization of the species of the Tetrahymena pyriformis complex. Trans. Am. microsc. Soc. 95, 664-682. NILSSON, J. R. & WILLIAMS, N. E. (1966). An electron microscope study of the oral apparatus of 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 a Tetrahymena mutant. Expl Cell Res. go, 345-356. PIPERNO, G., HUANG, B. & LUCK, D. J. L. (1977). Two-dimensional analysis of flagellar proteins from 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 of the 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 forming food vacuole. J. Ultrastruct. Res. 66, 132-150. VAUDAUX, P. (1976). Isolation and identification of specific cortical proteins in Tetrahymena pyriformis 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)