In autotrophically grown ChloreUa.cells, glucose induces a hexose transport system but, at the same time, the synthesis of two amino acid transport systems is also induced. Thus, the rates of uptake of glycine, L-alanine, L-proline, and L-serine, all of which compete with each other for entry into the cells, increase more than 1004fold when the algae are pretreated with glucose. The rates of L-arginine and L-lysine uptake increase by a factor of 25 to 50. The accumulation of proline and arginine within the cells amounts to 200-and 600-fold, respectively. Glucose does not cause the positive effect on. amino acid uptake by serving as metabolic substrate because the nonmetabolizable 6-deoxyglucose also acts as inducer. Cycloheximide prevents the induction. The induced transport system for the four neutral amino acids has a turnover with a half-life of 7 hr, which corresponds closely to the half-life of the hexose transport system. The transport system for the basic amino acids, on the other hand, disappears with a half-life of 25 hr.Since the classical work of Rickenberg et al. (1) on the induction of the lac. permease in Escherichia coli, many inducible transport systems have been described for bacteria (2-4). In a few cases this phenomenon has also been observed for eukaryotic cells (5, 6). Wherever induction has been found, however, the inducing substrates were either transport substrates and analogues ofthese or, like allo-lactose (7), a physiological substance closely related to the transport substrate.It has been surprising, therefore, to find that glucose and nonmetabolizable glucose analogues act as inducers for two specific amino acid uptake systems in the unicellular alga Chlorella vulgaris. This autotrophically growing alga rapidly takes up various hexoses once a specific hexose transport system is induced by monosaccharides (6, 8). This transport ofhexoses is an active one and proceeds as proton symport (9, 10). This paper reports that Chlorella induced for sugar transport also shows an uptake rate for basic and neutral amino acids that is increased more than 100-fold compared to cells not pretreated with hexoses. MATERIALS AND METHODSThe strain of C. vulgaris and the growth conditions were as described (8).]alanine, and L-['4C]proline were obtained from New England Nuclear; all other radioactive L amino acids were a gift of A. Bock. 6-Deoxyglucose was tritiated by Amersham. Cycloheximide and the nonradioactive amino acids were obtained from Serva (Heidelberg, Federal Republic of Germany), and 6-deoxyglucose was from Koch-Light (Colnbrook, England).Uptake ofAmino Acids. The screening for amino acid uptake that might be stimulated by glucose was performed with 0.1 mM labeled amino acid in an algal suspension of50 t1u ofpacked cells per ml in the presence or absence of 15 mM glucose. The disappearance of radioactivity from the medium was determined by centrifugation of the cells in intervals (2 or 5 min) and measurement of residual radioactivity in the medium. When the uptake of an amino acid was...

The substrate specificity of the glucose-proton symport system was studied to gain information about the spatial relationship between the binding sites for glucose and proton. Charged glucose analogues such as amino sugars or sugar acids were not transported by the uptake system, with the exception of 2-amino-2-deoxy-~-glucose. This glucosamine was taken up in the charged form in uniport mechanism, i.e. without symport of proton. This result was interpreted to mean that the proton-binding site of the symport system is close to the hydroxyl at carbon 2 of glucose.This interpretation was strengthened by the following facts: The steric position of hydroxyl groups at carbons 1, 2 or 3 of glucose were especially important for efficient transport. 0-Methylation was not tolerated at carbon 1, but it was tolerated at carbons 3,4 or 6.The stoichiometric flow of proton and sugar could be disturbed by removal of hydroxyl group at carbon 1 of glucose. The pH-dependence of sugar transport is sugar-specific, e.g. the amino group at carbon 2 of glucose improves transport at higher pH. The configuration at carbon 2 of glucose influences the specificity for the symported ion.It is concluded that the coupled flow of proton and glucose occurs by simultaneous coordinate movement of both in a transmembrane channel.Incubation of the green alga Chlorella vulgaris with glucose induces a hexose transport system which could be identified as a proton symport system [l, 21. A similar mechanism of transport energization works for nonelectrolyte transport in bacteria, fungi, higher plants and, with Na' instead of H', in animals [3 -61. The basic principles of substrate translocation might therefore be similar. In the past, studies were mostly concerned with the role of protonmotive force in driving nonelectrolyte accumulation. For Chlorella it was found that lack of protons (i.e. alkaline pH-value) drastically increases the K, value for hexose transport [7], while decrease of the membrane potential reduces the affinity of proton for hexose symport [7, 81. A schematic model has been devised where it was postulated that the transport protein has a gated pore with the binding site half-way through the membrane [9]. In this context it became interesting to elaborate the relative place and the structure of the binding site for hexose and for proton. There had been studies with Chlorella, which showed that no single hydroxyl group of the glucose molecule is essential for transport [lo, 111. Long before, careful studies of sugar specificity had been performed with red blood cells, small intestine and yeast [12-151. But all these studies have dealt with the sugar specificity per se and not with the relationship between binding of sugar and proton. The experimental approach for this report was to compare the specificity of the uptake system towards charged and uncharged substrates. The clarification of the interaction of sugar and proton binding could help to decide about several important points on the mechanism of transport such as: (a) is proton...

This study was undertaken in order to demonstrate the extent to which the activity of the plasmalemma H(+)-ATPase compensates for the charge and acidity flow caused by the sugar-proton symport in cells of chlorella vulgaris Beij.. Detailed analysis of H(+) and K(+) fluxes from and into the medium together with measurements of respiration, cytoplasmic pH, and cellular ATP-levels indicate three consecutive phases after the onset of H(+) symport. Phase 1 occurred immediately after addition of sugar, with an uptake of H(+) by the hexoseproton symport and charge compensation by K(+) loss from the cells and, to a smaller degree, by loss of another ion, probably a divalent cation. This phase coincided with strong membrane depolarization. Phase 2 started approximately 5 s after addition of sugar, when the acceleration of the H(+)-ATPase caused a slow-down of the K(+) efflux, a decrease in the cellular ATP level and an increase in respiration. The increased respiration was most probably responsible for a pronounced net acidification of the medium. This phase was inhibited in deuterium oxide. In phase 3, finally, a slow rate of net H(+) uptake and K(+) loss was established for several further minutes, together with a slight depolarization of the membrane. There was hardly any pH change in the cytoplasm, because the cytoplasmic buffering capacity was high enough to stabilize the pH for several minutes despite the net H(+) fluxes. The quantitative participation of the several phases of H(+) and K(+) flow depended on the pH of the medium, the ambient Ca(2+) concentration, and the metabolic fate of the transported sugar. The results indicate that the activity of the H(+)-ATPase never fully compensated for H(+) uptake by the sugar-symport system, because at least 10% of symport-caused charge inflow was compensated for by K(+) efflux. The restoration of pH in the cytoplasm and in the medium was probably achieved by metabolic reactions connected to increased glycolysis and respiration.

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