Dual capacity for nutrient uptake in Tetrahymena: V. utilization of amino acids and proteins

Many observations relevant to nutrient uptake have been made on Tetrahymena (Dunham & Kropp, 1973; Holz, 1973; Hutner, Baker, Frank & Cox, 1972; Nilsson, 1976; Rasmussen, 1976). Rates and kinetics of uptake of several compounds have generally been determined in non-nutritive buffers (Freeman & Moner, 1976; Hoffman & Rasmussen, 1972; Ling & Orias, 1974; Wolfe, 1975). Morphological observations have also been made on living cells with the light microscope, using coloured or refractile particles, or with the electron microscope on fixed cells (Chapman-Andresen & Nilsson, 1968; Nilsson, 1972, 1976). Because of the somewhat artificial experimental conditions and the variety of uptake routes available to nutrients (oral uptake system, carrier-mediated transport, possible extra-oral pinocytosis) the relevance of those observations to nutrient uptake and utilization in vivo has not been rigorously elucidated.

Recently, the isolation of a mutant with heat-sensitive food vacuole formation (Orias & Pollock, 1975) has provided a useful and versatile experimental tool with which to begin a rigorous dissection of the uptake routes which are used to satisfy nutritional requirements. Previous work using this mutant has shown that the oral uptake system is at least 20–100 times more efficient than any other uptake system of the cell for the utilization of Fe and Cu ions (Rasmussen & Orias, 1976) and the vitamin, folinic acid (Orias & Rasmussen, 1977). By contrast, the oral system appears to play a negligible role in the utilization of four other vitamins (nicotinic acid, pantothenate, riboflavin and pyridoxal; Orias & Rasmussen, 1977) and the nucleoside, guanosine (unpublished observations).

In the present study we have investigated the role of the oral system in the uptake of free amino acids -leucine (leu) and phenylalanine (phe) - and of the protein egg albumin, under conditions where the latter is asked to supply either all the ten required amino acids or a single one (phe or leu). By using the mutant at restrictive temperature, and thus eliminating the action of the oral uptake system (phagocytosis), it should be possible to reveal the function of any extra-oral, pinocytic system capable of nutritionally relevant protein uptake.

Our results have failed to provide any evidence that such an extra-oral system is involved in protein utilization. The importance of the oral uptake system for protein uptake has been revealed, and we have gained insights into ways in which this oral system contributes to protein utilization. The results also suggest that the oral uptake system plays a negligible role in free amino acid uptake at concentrations which are nutritionally adequate for the wild type cells.

Clone NP1 is derived from inbred strain D of Tetrahymena thermophila (previously known as T. pyriformis syngen 1 (Nanney & McCoy, 1976)). This mutant has a hcat-sensitivc development of its oral apparatus. At 37 °C it forms no food vacuoles detectable in the light microscope, but at 28 °C vacuoles are formed at normal rates (Orias & Pollock, 1975). The mutation(s) responsible for this phenotype is located in the macronucleus (Silberstein, Orias & Pollock, 1975). The mutant cells can be propagated for hundreds of fissions in the absence of food vacuoles by supplementing the nutrient media for wild type cells (2 % proteose peptone, or chemically defined medium, see below) with high concentrations of Fe³⁺, Cu³⁺ and folinic acid (Orias & Rasmussen, 1976). Clones D III (wild type inbred strain D, T. thermophila) and GL-8, T. pyriformis (Borden, Whitt & Nanney, 1973), both with normal capacity for food vacuole formation, were also studied.

A modification of the chemically defined medium of Holz, Erwin, Rosenbaum & Aaronson (1962) was used (Rasmussen & Modeweg-Hansen, 1973). This medium contains 17 amino acids (including the 10 required amino acids), the 7 required vitamins, 4 ribonucleosides, glucose, citrate and various inorganic salts. This medium was further supplemented with high concentrations of ferric chloride, copper sulphate and Ca-folinate (leucovorin, Lederle) to final concentrations of 1, 0·03 and 0·002 rnM, respectively, to allow adequate uptake of these compounds independent of food vacuole formation (Orias & Rasmussen, 1976). Phenylalanine or leucine (required amino acids) or the whole complement of amino acids was omitted from the media as desired.

Egg albumin (Sigma no. A5503) was dissolved in distilled water (10 × final concentration) and sterilized by filtration through a 0·22-μm pore size Millipore filter. Aliquots were placed in a boiling waterbath for 5 min and a fine particulate protein coagulate was formed. Dissolved or denatured protein was added to culture media as required.

The routine maintenance of NP1 and the wild type strains has been previously described (Orias & Pollock, 1975).

Experimental cultures were grown without shaking in 10-ml portions of the chemically defined medium in 125 ml-capacity, screw-cap Erlenmayer flasks. They were inoculated with an initial density of 5000 cells/ml, using cells grown for at least one transfer under the conditions being tested. On subsequent days cell concentrations were determined in aseptically removed samples. Cell concentrations were determined in a counting chamber if the medium contained egg albumin, or in a calibrated electronic cell counter otherwise.

Early experiments done to identify any additional nutritional requirements of mutant NP1 due to growth without food vacuoles indicated that the defined medium contains non-limiting amounts of free amino acids (Rasmussen & Orias, unpublished observations). Those experiments thus failed to show any need of the oral system for free amino acid uptake.

A more discriminating test of differences in amino acid utilization in the presence and absence of food vacuoles is to compare the wild type and mutant strains at concentrations where one amino acid becomes growth limiting. Accordingly, we have measured growth rates of both strains in media containing an excess of all required nutrients with the exception of one amino acid (phenylalanine or leucine), whose concentration in the medium was varied (Fig. 1). The identity of the growth response of both strains indicates that, from a nutritional standpoint, the oral uptake system plays a negligible role in the uptake of phenylalanine and leucine in their free form. These results agree with the failure of amino acid supplementation to improve the growth rate of NPi and with direct measurements which show that the oral uptake system contributes negligibly to total phenylalanine uptake (Ling & Orias, 1974).

The availability of a defined medium in which NPi can be grown without vacuoles, and the finding that the oral uptake system is unnecessary for free amino acid uptake, provide a useful experimental system in which to investigate the routes of protein utilization as an amino acid source. Specifically, what role does the oral system play in protein utilization? In the experiments described below we looked for the availability of egg albumin to supply either (a) all the required amino acids, or (b) a single required amino acid (phenylalanine or leucine).

Table 1 shows the results of an experiment in which 0·1 % egg albumin, either in native or heat-denatured form, was supplied as a source of all the required amino acids. These data allow the following conclusions: (a) 0·1% heat-denatured egg albumin is a nutritionally complete and quantitatively sufficient source of required amino acids; (b) utilization of this supply requires the oral uptake system, since egg albumin cannot replace the required amino acids for NP1 under conditions where it cannot form food vacuoles; (c) denatured (particulate) egg albumin can be utilized with much greater efficiency than native (soluble) egg albumin by the wild type, phagocytosis-competent cells.

The failure of wild type (or mutant) cells to grow on soluble 0·1% egg albumin cannot be attributed to the presence of a heat-labile inhibitor, since good growth is observed when soluble egg albumin and amino acids are added together. Similarly, the failure of NP1 cells to grow in denatured egg albumin cannot be explained by assuming that some required nutrient (other than an amino acid) is adsorbing to the egg albumin and thus becoming unavailable to the NP1 cells, because these cells grow well when both denatured egg albumin and amino acids are added together.

The above experiments were repeated under conditions where the egg albumin was the sole supply of the required amino acid phenylalanine. The results (Table 2) are essentially the same as when egg albumin must supply all the required amino acids (Table 1) or leucine alone (data not shown).

Why does food vacuole formation allow the utilization of 0·1% denatured egg albumin and not of 0·1% native egg albumin? Two possibilities can be considered: (1) the oral system can concentrate particulate egg albumin much more efficiently than soluble albumin, and (2) denatured egg albumin is much more susceptible to digestion by proteases in the food vacuole than native egg albumin. In order to investigate these possibilities, experiments using much higher concentrations of soluble egg albumin were done. It can be seen from the results (Table 3) that soluble egg albumin can be utilized as an amino acid source if the concentration is sufficiently high (at least 1 %). Furthermore, this utilization also requires the oral uptake system, since it is not observed in the NP1 mutant in the absence of food vacuole formation. These results suggest that when the cells are supplied 0·1% native albumin, the limiting factor is the amount of substrate (egg albumin) and not proteolytic (digestive) activity. Thus, at a 0·1% concentration of egg albumin, the food vacuole system can concentrate nutritionally adequate amounts of the protein in particulate form, but not in its native, dissolved form. The results strongly suggest the lack of any extravacuolar route of egg albumin utilization as an amino acid source.

Attempts to extend these experiments to other proteins have not yielded meaningful results, because growth in the presence of the denatured protein as amino acid source was either poor (bovine serum albumin) or absent (γ-globulin). In no case, however, was there any growth in the soluble (native) protein. Utilization of several denatured proteins and failure to utilize the same proteins in soluble form was independently reported earlier (Viswantha & Liener, 1956a).

Our results, indicate that, from a nutritional standpoint, the oral uptake system contributes negligibly (if at all) to phenylalanine and leucine uptake. This conclusion is strongly supported by direct measurements showing that carrier-mediated L-phenyl-alanine transmembrane transport proceeds with the same kinetics over a 200-fold concentration range, regardless of whether cells are capable of making food vacuoles or not (Ling & Orias, 1974). These results, taken together with the observation that further increases in amino acid concentration fail to stimulate growth of NP1 in the absence of food vacuoles, suggest that the Tetrahymena plasma membrane has efficient carrier-mediated systems for the uptake of all the required amino acids.

The ability to compare isogenic cells with and without a functional mouth and to control strictly the source of amino acids in the medium gives us a unique experimental system in which to dissect the routes of nutritionally meaningful protein uptake. The results show that egg albumin can supply the required amino acids only in cells with a functional mouth. The simplest interpretation of these results is that the mouth is the main or exclusive route of uptake of utilizable egg albumin. If other ways of taking up egg albumin exist, they are insufficient to fulfil nutritional needs for amino acids.

In a collaborative study with Dr Jytte Nilsson, we have observed small vesicles that incorporated ferritin during a 30-min exposure in NP1 cells growing in enriched 2% proteose at 37 °C (see discussion by Nilsson, 1977). Since these vesicles were not observed in the similarly treated (but food vacuole-forming) wild type control, it seems likely (but not yet proven) that the vesicles reflect a residual low level of endocytosis (micropinocytosis) occurring at the abnormally developed NP1 mouth. Nilsson has suggested that these pinocytic vesicles may account for the ability of strain NP1 to grow in proteose peptone without forming food vacuoles (Nilsson, 1977). Given the information presently available, we cannot agree with Nilsson’s suggestion. The diameter of the vesicles (one fifth of normal food vacuoles or less) and the lack of concentration of the ferritin in these vesicles suggest that NP1 can incorporate at most 0·1% (and very likely, much less) of the ferritin incorporated by the similarly treated wild type control. Thus, the results with ferritin uptake seem to us to confirm our conclusion that the mouth is essential for protein utilization. In view of both results, we are forced to assume that in proteose peptone at 37 °C, NP1 supplies its amino acid requirements mainly or exclusively by utilizing free amino acids and oligopeptides transported through carriers in the plasma membrane.

Our experiments with dissolved egg albumin give insights into the physiological mechanisms of the oral system in protein uptake. We estimate that the oral system can concentrate 1 % dissolved protein by at least one order of magnitude with respect to the growth medium, based on the following values determined under the appropriate conditions. The total food vacuole volume formed/generation is 10000 μm³ (newly formed food vacuole diameter, 6 μm; number of food vacuoles formed/cell cycle, 85). This volume amounts to about 50% of the mean cell volume (22000 μm³ as determined by cytokrit, corrected for intercellular space). The cell protein is 5% of the cell weight (protein is about 50% dry weight (Leick, 1967), dry weight is 10% of wet weight) and the phenylalanine concentrations in Tetrahymena protein (about 5%; Wu & Hogg, 1952) and in egg albumin (5·5%; Virtanan, Laine & Toivonen, 1940) are roughly equal. If we assume that the total food vacuole volume per cell generation (5°% of the average volume) packs enough protein (5%) to supply the cell’s need for the essential amino acid phenylalanine, then the protein concentration in the food vacuoles must be at least 10%, or 10-fold higher than the external medium (more, if not all phenylalanine is utilized). This estimate is comparable to that of Ricketts & Rappitt(1975a), based on incorporation of dissolved radioactive bovine serum albumin into Tetrahymena cells.

Our finding that wild type Tetrahymena requires at least a 1 % concentration of native egg albumin for growth (Table 3), is consistent with Viswanatha & Liener’s (1956a) observation that native egg albumin (maximum concentration used: 0·13%) did not support growth in these cells. These authors were the first to note that Tetrahymena utilize heat-treated better than native proteins.

What mechanism is responsible for concentrating dissolved protein in the newly formed food vacuole? The trapping of dissolved egg albumin cannot be easily attributed to any direct sweeping action of the oral membranelies (Rasmussen, Buhse & Groh, 1975). The necessity to postulate a ‘glue’ seems inescapable. As suggested by Nilsson (1976), the glue could be the mucocyst content. Mucocysts are exocytic vesicles distributed throughout the plasma membrane. Their content, upon release to the medium, could bind nutrients and be ingested, as a complex, through the mouth (Nilsson, 1976). Although ingestion of alcian blue bound to mucus has been seen (Nilsson, 1972), the extent of the contribution of mucus binding to nutrient uptake remains untested. Alternatively, this ‘glue’ could be a membrane-bound substance lining the incipient food vacuole. The same mechanism could trap the aggregates present in the denatured agg albumin. A source of this protein-binding material could be the small diskoidal vesicles which, in Paramecium, are incorporated, by exocytosis, into the nascent food vacuole membrane (Allen, 1974). Whatever the nature of the binding material, it plays no detectable role in free amino acid uptake at concentrations that saturate the nutritional requirements of the cell, according to the work reported here.

Extra-oral micropinocytosis (Allen, 1967) has often been considered as a third route of normal uptake of nutrients in Tetrahymena (in addition to food vacuole formation and carrier-mediated transport through the plasma membrane). The para-somal sacs, which are permanent invaginations of the plasma membrane arranged in regular spatial relationship to each kinetosome of the somatic ciliature, have been suggested as a possible site of such micropinocytosis. Indeed, in the peritrich ciliate Trichodinopsis (Noirot-Timothée, 1968) and in the suctorian Tokophrya (Rudzinska, 1977a, b), evidence that micropinocytosis occurs in the parasomal sacs has been presented, although a rigorous demonstration of an inward movement of material is lacking. It is not yet known what the physiological significance of this micropinocytosis is, in nutrition or otherwise. Similar vesicles have been observed in Tetrahymena in the vicinity of the parasomal sacs (Allen, 1967; Williams & Luft, 1968). It remains to be tested by electron-dense labelling whether or not these vesicles are indeed formed by endocytosis at the parasomal sac membrane.

Work possibly relevant to the occurrence of micropinocytosis in Tetrahymena has been carried out by Ricketts & Rappitt (1975b). They measured the amount of radioactive egg albumin that became resistant to washing, during a 100-min period at 20 °C in the presence or absence of cytochalasin B and/or cycloheximide, and correlated it with the number of food vacuoles present at the end of the treatment. (Both of these drugs inhibit food vacuole formation, among other effects; Frankel, 1970; Nilsson, 1977; Nilsson, Ricketts & Zeuthen, 1973; Ricketts & Rappitt, 1975 a, b). However, no direct measurements of micropinocytosis were made, nor was there any independent and rigorous measurement of the amount of label incorporated into food vacuoles or of the total brought to the inside of the cell (as opposed to binding tightly to the cell surface). These experiments are therefore inconclusive with regard to the occurrence and extent of micropinocytosis. The authors concluded nevertheless that, if it occurs, it plays a ‘much less important role’ in nutrition than food vacuole formation. Nilsson, on the other hand, has reexamined Ricketts & Rappitt’s data and concludes that micropinocytosis in cytochalasin-inhibited cells accounts for 50% of the egg albumin uptake in control cells (Nilsson, 1977). She ascribes this uptake to an observed residual micropinocytosis believed to take place at the mouth in cytochalasin-B-inhibited cells, and which is not observed in wild type cells. If Nilsson’s suggestion is correct, the small size of the pinocytic vesicles demands that they enclose highly concentrated protein and that they be formed at an exceedingly high rate. The plausibility of such a high residual micropinocytic uptake rate remains to be checked, either nutritionally, e.g. by growth in a defined medium where endocytosis is absolutely required (as done here), or by EM observations, e.g. ferritin uptake.

In summary, then, residual micropinocytosis has been reported in the developmentally defective NP1 mouth and in the cytochalasin-inhibited wild type mouth. We believe that the former does not play a nutritionally significant role in protein uptake and utilization, while the role of the latter remains untested. There are no rigorous EM observations or measurements in Tetrahymena that demonstrate the occurrence of micropinocytosis at locations other than the mouth. Even if such mechanisms were demonstrated, our finding that egg albumin utilization requires the oral system suggests that such putative micropinocytic mechanisms play a nutritionally insignificant role in protein uptake, and any possible function should be sought elsewhere.

Tetrahymena cells release proteases into the growth medium but these proteases are unable to hydrolyse egg albumin extensively (Viswantha & Liener, 19566). The failure of the wild type strain to grow in less than 1 % native egg albumin is consistent with these conclusions. The functional contributions of these proteases, therefore, remain unknown.

The finding that the utilization of egg albumin depends mainly or exclusively on food vacuole formation provides a basis for isolating mutants which have genetic defects in this uptake mechanism, not only at the level of food vacuole formation (like NP1, or other mutants with similar phenotype recently isolated; Suhr-Jessen, 1977; Suhr-Jessen & Orias, 1979), but also at the level of digestion by lysosomal enzymes and uptake through the food vacuole membrane. The most recent advances in methods of mutant induction and isolation in Tetrahymena thermophila (Bruns & Sanford, 1978; Orias & Bruns, 1976; Orias, Hamilton & Flacks, 1978) hold the promise of a productive application of this knowledge to a genetic dissection of lysosomal biology.

We thank Birthe Dohn and Judy Orias for excellent technical assistance, Cicily Chapman-Andresen, H. Holter, G. G. Holz, Jr, M. Müller, K. M. Moller, Jytte R. Nilsson, P. B. Suhr-Jessen and E. Zeuthen for useful discussions and/or critical reviews of the manuscript, and Miriam Flacks for editorial assistance. E.O. gratefully acknowledges sabbatical leave and research support from the Carlsberg Foundation and NIH Grant GM21067.