Calcium Modulation of Polyamine Transport Is Lost in a Putrescine-Sensitive Mutant of Neurospora crassa’

Putrescine transport in Neurospora is saturable and concentrative in dilute buffers, but in the growth medium putrescine simply equilibrates across the cell membrane. We describe a mutant, puu-1, that can concentrate putrescine from the growth medium because the polyamine transport system has lost its normal sensitivity to Ca2+. The wild type closely resembles the mutant if it is washed with citrate and ethylene glycol bis(&aminoethyl ether)N,N’-tetraacetic acid. The mutant phenotype also appears in the wild type after treatment with cycloheximide. The results suggest that putrescine uptake is normally regulated by an unstable Ca2+-binding protein that restricts polyamine uptake. This protein is evidently distinct from the polyamine-binding function for uptake, which is normal in mutant and in cycloheximide-treated wild type cells. The puu-1 mutation, stripping of Ca2+, and cycloheximide treatment all cause an impairment of amino acid transport, indicating that other membrane transport functions rely upon the product of the puu-l+ gene. Preliminary evidence suggests that the putrescine carrier is not the

Putrescine transport in Neurospora is saturable and concentrative in dilute buffers, but in the growth medium putrescine simply equilibrates across the cell membrane. We describe a mutant, puu-1, that can concentrate putrescine from the growth medium because the polyamine transport system has lost its normal sensitivity to Ca2+. The wild type closely resembles the mutant if it is washed with citrate and ethylene glycol bis(&aminoethyl ether)N,N'-tetraacetic acid. The mutant phenotype also appears in the wild type after treatment with cycloheximide. The results suggest that putrescine uptake is normally regulated by an unstable Ca2+-binding protein that restricts polyamine uptake. This protein is evidently distinct from the polyamine-binding function for uptake, which is normal in mutant and in cycloheximide-treated wild type cells. The puu-1 mutation, stripping of Ca2+, and cycloheximide treatment all cause an impairment of amino acid transport, indicating that other membrane transport functions rely upon the product of the puu-l+ gene. Preliminary evidence suggests that the putrescine carrier is not the Ca'+-sensitive, low-affinity K+-transport system, but K+ efflux does accompany putrescine uptake. 0 1991 Academic Press, Inc. Most organisms take up polyamines (putrescine, spermidine, and spermine) by an active transport system, despite their ability to make these compounds internally. The system may allow adjustments to the polyamine pools of cells of multicellular organisms; it may serve polyamine catabolic pathways; and it may even allow accumulation of putrescine as an osmoticum.
Neurospora crassa makes polyamines, but does not use them as catabolic substrates. The organism has a polyamine transport system that takes up all three natural polyamines with moderate affinity (Km's: putrescine = 600 PM; spermidine = 240 PM; spermine = 70 PM) from a dilute buffer containing 20 mM Na+ (1). In the growth medium, however, this system is inhibited by monovalent cations and by Ca2+, and all transport of polyamines takes place by way of a nonsaturable, diffusional system that equilibrates polyamines across the cell membrane (1,2). The concentrative uptake system appears only in Ca'+free buffers having low monovalent cation concentrations. We have in fact questioned whether the natural substrates of the system include the polyamines (1).
We describe here a mutant of N. crassa, puu-1, able to concentrate polyamines from low concentrations in the medium, owing to a loss of inhibition of the polyamine uptake system by cell-bound Ca2+. The strain is intoxicated by polyamines in concentrations to which the wild type is indifferent. The results suggest that a component of the cell membrane protects N. crassa from absorbing toxic levels of polyamines-and perhaps other cationsfrom the environment. The accompanying paper (3) defines the discretionary capacity of N. crassa for polyamines and its intracellular location, using the puu-1 and another putrescine-accumulating strain. Medium (2 ml) from a culture was applied to the column, then the column was washed with 15 ml 1.5 N HCl, and 6 ml of 3.25 N HCl. Putrescine was then eluted in 6 ml 3.5 N HCl; following this, spermidine was eluted in 6 ml 6 N HCl. Spermine (2 nmol) (Table I).

MATERIALS AND METHODS
min, without subtracting the diffusional component.
Additional mutants with the puu-1 growth phenotype We used doubling time (in minutes) and polyamine pool sizes (see were selected after mutagenesis. The severity of the pu-Results) to calculate steady-state parameters of influx and efflux. The rationale was that of Karlin et al. (7), based simply on a growth equation where A, and A,, are the values at the end and beginning of an interval (1 min); t is time in minutes; and k is the growth constant (In 2 divided by doubling time).

TABLE I
Progeny of the Cross cot-l puu-1 arg+ X cot+ PULL+ arg-2 Uptake of i4C-labeled amino acids of known specific radioactivities was measured by methods similar to those used for polyamine uptake. Activities of three amino acid uptake systems were measured at 25'C for 2 min. The general system substrate was 1 mM ["Claminoisobutyric acid (8); the neutral system was measured with 1 mM [3H]leucine in the presence of 1 mM arginine (9); and the basic system was measured with 0.1 mM ["Clarginine in the presence of 0.1 mM leucine (10). K+, Na+, Ca*+, and Mg2+ were determined by atomic absorption spectroscopy as described previously (11). Polyamine extraction and determination by high performance liquid chromatography were done as described previously (12). trescine uptake or inhibition phenotypes varied widely, and most suffered growth or morphological defects that made them difficult to work with. None was allelic topuu-1. Reversions of the puu-1 mutant were similarly variable. Almost all were due to unlinked suppressor mutations, and none lacked the polyamine transport system. Limited genetic tests showed many reversion events to be nonallelic to one another. Because of the pleiotropy of puu-1 and the diversity of mutations that duplicated or reversed its effect, further genetic studies were deferred in favor of characterizing the original mutant in detail. A heterokaryon containing arg-2 puu-1 inl+ and arg+ puu+ in1 nuclei grew well in minimal medium supplemented with 5 mM putrescine. This indicated that the puu-1 mutation was recessive to its wild type allele and thus represented a deficiency or loss of function.

Altered Transport Characteristics of the puu-1 Mutant
Wild type Neurospora cannot concentrate polyamines from Vogel's medium, because the saturable polyamine transport system is inhibited (1,2). Putrescine and spermidine equilibrate across the cell membrane by a diffusional mechanism (2). In the same medium, the puu-1 strain displays saturable uptake of putrescine and spermidine ( Fig. 1). This accounts for the origin of the puu-1 mutation by the selection method we used. The final slopes of the lines in Fig. 1 revealed that the strains shared the diffusional component of uptake. The apparent Km's of the puu-1 strain were about 3 mM for both substrates under these conditions. After a wash and resuspension in Mops buffer (20 mM Na+), both strains displayed saturable polyamine uptake, with normal K,,,'s for putrescine (0.3-0.7 mM) and spermidine (0.25 mM) (1); the Km's were lower in buffer owing to lack of competitive inhibition by salts in the medium. The V,,, of uptake bypuu-1 strains in Mops buffer was 2.5 to 8 times greater than that of the wild type. The putrescine uptake rate and K,,, of both strains were insensitive to pH variation between 5.2 and 8.2 (data not shown).
The putrescine pool of both Puu+ and Puu-strains grown in minimal medium is approximately 0.8 to 1.0 nmol per milligram dry weight (1,3). The wild type, grown in the presence of 1 mM putrescine, has a putrescine pool of about 2 to 3 nmol per milligram, or approximately 1 mM in cell water [2.5 ~1 per milligram dry weight (14)]. This bears out our previous finding that putrescine equilibrates diffusionally across the cell membrane (2). However, in 1 mM putrescine, the puu-1 strain contains approximately 120 nmol putrescine per milligram, nominally about 48 mM, indicating considerable concentrative transport. The molar value is belied by the fact that most of the putrescine is in the vacuole (3) strains in their ability to accumulate putrescine is roughly correlated with the V,,, of the strains' saturable uptake systems (see below). During steady-state growth of either strain in putrestine, the influx rate should be the same as the rate of efflux plus whatever is required to maintain polyamine pools during growth. If the high pool of putrescine inpuu-1 were primarily the result of impaired efflux, influx rates at steady-state would be normal or low. If the high pool was owing to a higher rate of influx, the influx rate at steady-state should remain high. A test of these expectations showed that pm-1 uptake at steady-state was much higher than that of the wild type, indicating that influx is altered in the mutant (Fig. 2).
We also calculated the amount of putrescine uptake required to maintain the putrescine and spermidine pools of the two strains during growth in 1 mM putrescine (3), assuming no contribution from biosynthesis. This showed that the measured rate of uptake at steady state (0.11 and 0.5 nmol per minute per milligram for wild type and puu-1, respectively, from Fig. 2) roughly approximated the need (0.14 and 0.6 nmol per minute per milligram, respectively). Only a very small efflux was expected from wild type, and, indeed, little efhux from wild type can be seen upon transfer from 1 mM putrescine to minimal medium. A small, but definite efflux (0.1 nmol per minute per milligram) was actually seen for puu-1 in a parallel experiment, in keeping with its higher intracellular putrescine concentration. Spermidine et&x was not detected in either strain, either in the parent culture or in the minimal medium to which it was transferred. These calculations and observations show that the primary effect of the puu-1 mutation is on influx, rather than efllux of putrescine.
The reason that putrescine efflux from puu-1 was not much greater is that excess intracellular putrescine under these conditions is largely vacuolar (3). In the accompanying paper, we show the vacuolar accumulation of putrescine inpuu-1 is the result, not the cause, of putrescine accumulation in the cell itself.
Differential Sensitivity of Wild Type and puu-1 to Calcium Most cations of the medium inhibit putrescine uptake, the effect of Ca2+ being the most severe (1). Unlike omissions of other cations, omission of Ca2+ from the medium led to an increased putrescine uptake rate in the wild type and a slightly decreased uptake rate in puu-1 (Fig. 3). Omission of Ca'+ also allowed the wild type to accumulate a substantial putrescine pool from media containing 1 mM of the diamine (Fig. 4). Varying the Ca2+ concentration of the medium reveals an extreme difference in the sensitivities of wild type andpuu-1 cells (Fig. 4). The data suggested that the competitive inhibition of putrescine uptake by Ca2+ previously described for wild type (1) might be less strong in puu-1.
This idea was tested by testing washed cells of the two strains in Mops buffer for the sensitivity of putrescine uptake to Ca2+. Unexpectedly, putrescine uptake was found to be equally sensitive in the two strains (Fig. 5), although they had different V,,,,, for transport.
The discrepancy between the strains' sensitivities to Ca2+ in Mops buffer (Fig. 5) and in growth medium (Figs. 3 and 4) was traced to citrate, a metal chelator present at 8 mM in the medium (Table II, Experiment  I). If Ca2+ inhibited putrescine uptake solely by competitive inhibition (Fig. 5), citrate should mitigate the inhibition in both strains to the same degree by reducing the concentration of the free metal. As noted, this was not the case. The differential effect of citrate on the two strains with respect to Ca2' inhibition therefore suggested (i) that Ca2+ had a second inhibitory action on wild type; (ii) that a high-affinity Ca2+ binding site was involved, such that citrate was only weakly effective against added Ca2+; and (iii) the puu-1 mutant lacked sensitivity to this action of Ca2+, which accounts for its ability to concentrate putrescine from the growth medium. Further experiments supported this scenario.

Cell-Bound Calcium Inhibits Putrescine Uptake Only in Wild Type
Naa citrate stimulated putrescine uptake by the wild type, even in the absence of Ca2+ (Table II). In the same experiment, Nas citrate inhibited uptake bypuu-1, an effect exerted by the Na+ counterion (NaCl had a similar effect). The stimulation of the wild type system by citrate (actually minimized by the Na+ counterion) suggested that transport in the wild type was controlled by a chelatable factor firmly bound to the cell surface. Ca2' added back in the presence of citrate inhibited putrescine uptake only in the wild type (Table II) In this experiment the strains were grown in the normal or Car+-deficient medium; virtually identical data came from cells grown in normal medium and merely tested under the two conditions. Pools of polyamines in the two strains were normal, and not affected by growth in the absence of Car+. Ca2+ omission from the medium, suggest that the citratechelatable factor originally bound to wild-type cells was Ca2+. Using puu-1 and wild type strains, we compared the effect of a brief rinse of cells in 1 mM EGTA with a 2 min preincubation in 10 mM Nas citrate plus 10 mM EGTA, each followed by a wash in Mops buffer (Fig. 6). The brief rinse left the two strains with different uptake rates, and with opposite responses to added citrate (Fig.  6A, 0 Ca2+), as seen before. The effect of Ca2+ bore out previous experience: Ca2+ inhibited both strains similarly, but citrate mitigated this effect only in the mutant (Fig.  6A). The strong chelator wash, however, rendered the wild type phenotype almost identical to that of the mutant (Fig. 6B). At 0 Cazf, the putrescine uptake rates were the same; Na, citrate inhibited the strains similarly (via the Na+ counterion); and now added Ca2+ inhibited neither strain when citrate was present.
This experiment illustrates the membrane-bound nature of the chelatable element and introduces another phenomenon: the lability of the Ca2+-binding site. First, briefly rinsed wild type cells retain their low rate of transport, suggesting that the inhibitory Ca2+ is still bound to the cells. The further inhibition of transport by added Ca2+ is owing to the competitive inhibition common to the two strains (Fig. 5). Briefly rinsed wild type cells, tested in the presence of 10 mM citrate and the absence of Ca'+, release cell-bound Ca2+, and have an increased rate of uptake (Fig. 6A). However, when more than 0.5 mM Ca2' is also present, it inhibits, owing to the relative ineffectiveness of citrate in protecting the high-affinity binding site from Ca2+. The latter site must be missing from puu-1, inasmuch as Ca2+ inhibits the mutant only weakly in the parallel test (Fig. 6A).
Turning to cells washed in strong chelator, freed of the wash medium and tested in Mops buffer, we found the wild type relieved of its endogenous inhibitor, and in the absence of Ca2+ or citrate, its uptake rate is even higher than that of puu-1. Moreover, it can no longer be inhibited by Ca2+ in the presence of citrate. The latter finding suggests that once Ca2+ is definitively removed, its site of action is lost rapidly from wild type cells.
The calcium requirement for growth in stationary liquid culture was similar in the wild type andpuu-1, and both strains achieved 18 to 24% of their maximal weight in Ca2+-free medium containing 10 mM EGTA. Putrescine did not stimulate growth of Ca2+-free cultures. The Ca2+ content of puu-1 mycelia grown in minimal medium was somewhat higher than in the wild type, but most of the excess could be removed with a brief rinse in 1 mM EGTA. About 10-13s of the intracellular Ca2+ of both strains was found in vacuoles. The growth of both strains was inhibited similarly by Verapamil, a Ca2+-blocker; trifluoperazine, which inhibits Ca2+-binding proteins; and the Ca2+ ionophore A23187, indicating no fundamental dif-[Ca*+ I, yM Inset: reciprocal of uptake rate vs inhibitor concentration.

DAVIS
ET AL. ference between the two strains in Ca2+ transport or metabolism.

Na+-K+ Exchange Remains Ca2f-Sensitive in the puu-1 Mutant
The exchange flux of K+, and the exchange of K+, Na+, and H+ is severely inhibited by Ca2+, and Ca2+ therefore contributes to the ability of N. crassa to maintain a high membrane potential (15). Because a number of unusual amines (e.g., Tris, choline, imidazole, triethylamine) are reported to stimulate K+ efflux in Ca2+-free buffers (15), we considered that the K+ carrier might actually transport all of them, as well as putrescine. If this were the case, putrescine should not only promote K+ efflux, but we would expect K+ efflux promoted by Na+ in the puu-1 mutant to be Ca2+-insensitive. Initial experiments showed that indeed substantial K+ efllux followed the addition of Other Characteristics of the puu-1 Mutant The puu-1 strain is more sensitive than the wild type to paraquat, a poison that enters via the polyamine transport system in some mammalian cells (16). The wild type was unaffected by 10 PM paraquat, while puu-1 was inhibited 64%. La3+ (10m3 M) inhibited growth of the puu-1 mutant about 40%, whereas the wild type was not affected. We do not know whether La3+ is transported into cells, nor whether it competes with polyamines in the transport process.
Thepuu-1 mutant displays only 20 to 50% of the normal activity of the general amino acid uptake system and a and to CaCl, after mild and strong chelator treatments. A, mycelia rinsed briefly in 1 mM EGTA before testing in Mops buffer (10 mM Na+) with additives.
B, mycelia incubated 2 min in 10 mM EGTA and 10 mM citrate, followed by thorough wash in Mops buffer before test of uptake in Mops (10 mM Na+). Open symbols, wild type; closed symbols, puu-I. Circles, no citrate; squares, 10 mM Na, citrate during uptake test. The figures for 0 Ca*' in (A) and (B) are also reported in Table II, Experiment II. less reproducible deficit in amino acid uptake by the basic and neutral systems. Amino acids do not compete seriously with polyamine transport by wild type (1). None of the mutations of the three amino acid uptake systems (pmn, pmg, or pmb) affects putrescine uptake, and none of them is closely linked to puu-1. Stripping of Ca*+ from wild type cells by washing them with 10 mM citrate + 10 mM EGTA leads to a severe deficit (82%) in general amino acid transport when compared with cells rinsed in 1 mM EGTA. The data suggest that puu-1 is pleiotropic, and not specifically affected in the transport of polyamines. However, it should be stressed that the mutation (and Ca'+-depletion of the wild type) affects polyamine and amino acid transport in opposite directions, The Effect of Cycloheximide on Putrescine Transport Cycloheximide, added to wild type cultures in amounts sufficient to stop protein synthesis, induced the ability to concentrate putrescine from the growth medium. By 60 min after cycloheximide addition, the cells resembledpuu-1 (Fig. 7). Tests of the concentration-dependence of polyamine transport in cycloheximide-treated cells in Vogel's medium showed that they had gained activity for the saturable system (data not shown). The same treatment of puu-1 cells, already displaying the saturable transport system in the growth medium, left their polyamine transport rate (Fig. 7) and pools unaltered. The effect of cycloheximide was through its effect on protein synthesis: mutants (cyh-1 and cyh-2) resistant to cycloheximide by virtue of ribosomal alterations were unaffected by the drug (data not shown). After 10 min incubation in cycloheximide, the general amino acid uptake system of the wild type had declined to 54% of normal; the puu-1 mutant was not tested.

DISCUSSION
The puu-1 mutation of N. crassa renders polyamine uptake insensitive to cell-bound Ca2+ and thus imparts to ceils the ability to concentrate polyamines from the Ca2+-containing growth medium. The wild type strain, by contrast, can only equilibrate polyamines across the cell membrane by a nonsaturable process. The mutant differs in having a higher rate of polyamine uptake, not a higher affinity for substrate. Saturable putrescine uptake is accompanied by efflux of Kf, and both fluxes respond to the effects of cell-bound Ca2+ (lower) and to the puu-1 mutation (higher). The puu-1 mutant is partially deficient in the activity of the general amino acid uptake system and perhaps of the neutral and basic systems as well. On minimal medium, however, Na+, Kf, Ca'+, and Mg2+ contents of puu-1 cells are normal, as are the endogenously synthesized arginine and polyamine pools (3).
In some respects, the puu-1 mutation resembles a mutation, spsA1, of Aspergillus nidulans, roughly characterized by Spathas et al. (17). The spsA1 mutation makes the fungus sensitive to higher concentrations of spermidine, the carrier for which appears to be distinct from the putrescine carrier (18). Putrescine, in fact, did not inhibit the spsA1 mutant.
Ca2+ has two inhibitory actions on polyamine uptake by the wild type. The first is competitive inhibition of uptake (1). The puu-1 mutant is as sensitive as the wild type to this action of Ca2+. The second inhibitory action of Ca2+ in the wild type was revealed by stripping the metal from cells by a strong chelator wash. After such a wash, the V,,, of the wild type polyamine uptake system increases to that of the mutant. cellular Ca2' content and localization, of the effect of Ca2+related antimetabolites, and of the growth requirement for Ca2+ reveal no fundamental derangement of Ca2' metabolism in the mutant. We therefore infer the existence of a cell-surface protein that, when bound to Ca2+, normally blocks polyamine uptake. This protein or function is labile, because sensitivity of polyamine uptake to Ca2+ is lost upon definitive removal of Ca2+, and upon incubating cells with cycloheximide.
The observation that cycloheximide treatment and the removal of Ca2+ do not affect polyamine uptake by the puu-1 mutant shows that these treatments target the same system affected by the puu-1 mutation.
Unlike polyamine transport in N. crassa, removal of membrane-bound Ca2+ impairs transport processes in many other organisms.
In fact, it impairs amino acid transport even in N. crassa. Calcium chelators partially inactivate the transferrin receptor of rabbit reticulocytes (19) and the uptake of y-aminobutryate in synaptosomes (20). Ca2+ depletion slows net uptake of K+ in corn root (21,22); it diminishes lysine, arginine, sulfate, glucose, malate, glycerate-3-P, and uracil transport in tobacco cells (23, 24). Smith (25) used many of the same treatments we have used in his study of Ca2+-stimulated serine uptake in tobacco cells. He proposed that Ca2+ allows cells to maintain a high membrane potential, upon which serine transport and retention depends. Cells lose transport activity in the presence of K+ (which depolarizes cells), but do so much more slowly if CaC12 is also present (25). Jones and Jennings (26) made the same general finding with respect to the growth of the fungus Dendryphiella salina. The point was made there that Ca2+ reversed Na+ inhibition of growth by preventing loss of internal K+.
Why, then, is polyamine transport in N. crassa uniquely stimulated, rather than inhibited, by removal of Ca2+ (and, by analogy, by the puu-1 mutation)?
Three component issues allow us to explore this matter: (i) What drives polyamine uptake? (ii) What is the polyamine carrier? (iii) What is the nature of the puu-1' product? Inorganic ion transport in N. crassa has been studied in great detail in the laboratories of C. L. Slayman and C. W. Slayman. The membrane potential, established through the activity of the plasma-membrane Hf-ATPase (27), is the energy source for most secondary transport (28). The membrane potential drives, via K+-Ht symport (29), the establishment of a substantial K+ gradient (30). (This requires extrusion of 2H+, mimicking a 1:l K'-H+ exchange.) If K+-replete cells are held in water, they retain Kt, but upon addition of Na+, they readily exchange internal K+ for the external cation (14). This has led the Slaymans (15) to analyze a fluent K+/Na+/H+ exchange flux characteristic of N. crassa, depending upon the ions present in the cell and in the external medium. Significantly, Ca2+ inhibits the K+/K+ exchange flux, but not the net K+ accumulation, about 80%) with a concomitant increase of membrane potential [ (31); summarized in Ref.
(29)]. In other words, Ca2+ makes the membrane less leaky, and energy otherwise dissipated is spared for improved transport of other solutes, as suggested above. A substantial net efflux of Kt occurs in our standard Na+-Mops buffer, which contains no added Ca2'. Na+ entry presumably balances Kt efflux to a large extent under this condition, as the Slayman laboratories have found (15). Addition of putrescine causes an even greater rate of K+ efllux in the puu-1 strain, concomitant with demonstrable entry of the amine. This behavior resembles previous observations that without Ca2', addition of imidazole, histidine, Tris, choline, ethanolamine, triethylamine, and NH: all caused loss of K+ and Na+ (15). Whether these compounds were taken up at the same time was not directly tested, but the membrane potential was not seriously affected (C. L. Slayman, personal communication).
If the effects of Ca2+ on putrescine uptake are opposite to those on amino acids and, in other organisms, most other solutes, we must consider the possibility that putrescine can become part of the exchange flux involving K+ and Na+. In this sense putrescine transport as we have measured it here would not be immediately dependent upon membrane potential, but more directly dependent upon the transmembrane gradient of K+. Energy is ultimately required to establish this gradient, but we have speculated previously that a residual Kt gradient may account for energy-independent putrescine uptake (1). We do not know the identity of the putrescine carrier. We feel that to N. crassa, putrescine is a peculiar substrate for transport into the cell. The fungus does not require external polyamines for growth, nor does it use them as C and N sources (1). The ability of N. crassa to take up polyamines is not an obvious advantage, and we have implied that the "polyamine transport system" may have evolved in relation to other needs entirely (1). Thus polyamine transport may be a gratuitous response to unusual circumstances-the absence of Ca2+ and the presence of polyamine.
It is unlikely that the "polyamine transport system" is one of the K+ carriers, of which there are high-and lowaffinity forms in N. crassa and in yeast (32,33). The data are too preliminary to exclude this possibility, but the puu-1 mutant does retain Ca2' sensitivity of K+ efIlux upon Na+ addition (as opposed to addition of putrescine). The fact that K+, Nat, and NH: all competitively inhibit putrescine transport; that both Kt and putrescine influx are inhibited by Ca2+, and that many amines, including putrescine, stimulate K+ efllux may indicate only an indirect coupling of the various transport systems, and independent modulation of these systems by Ca2+.
Cell-bound Ca2+ is a prime influence on the rate of putrescine entry. This leads to consideration of the last question: what is the puu-1 product? Impairment of the puu-1' product (by mutation, turnover, or removal of Ca2+) increases putrescine uptake activity. Absence or impairment of the puu-l+ product renders the organism vulnerable to polyamine intoxication (3). Our results suggest that the puu-1' product is a Ca2+-binding protein that imparts a beneficial impermeability of the plasma membrane to toxic materials (of which putrescine and La3+ are two). The puu-1 product also plays a significant role in the activity of amino acid and perhaps other transport systems. The protein could work by any of a number of means, from improving specificity of an unknown cation carrier by direct contact to changing the character of the bilayer in which this and other carriers are embedded. The pleiotropy of the puu-1 mutation suggests a large scope of involvement with cation transport. Defining the character of the puu-1 gene product will help us to discover its action, and perhaps the identity of the putrescine carrier and other carriers affected by the puu-1 mutation.