Removal Of Methionine Research Articles

Selective removal of N-terminal methionine from recombinant human growth hormone by an aminopeptidase isolated from Aspergillus flavus

An aminopeptidase was isolated from a soil fungus, which specifically cleaves the unnatural N-terminal methionine in recombinant human growth hormone. Reaction mixtures with different ratios of aminopeptidase to recombinant methionyl human growth hormone showed that the removal of N-terminal methionine was complete at 1:200 (w/w), and more than 90% complete at ratios up to 1:2000 (w/w) when incubated for 24 h at 37°C. The data indicate that the aminopeptidase we have purified can be used for the efficient conversion of unnatural recombinant proteins to their natural form.

Methionine aminopeptidase from the hyperthermophilic Archaeon Pyrococcus furiosus: molecular cloning and overexpression in Escherichia coli of the gene, and characteristics of the enzyme.

A gene for a methionine aminopeptidase (MAP; EC 3.4.11.18), which catalyzes the removal of amino-terminal methionine from the growing peptide chain on the ribosome, has been cloned from the hyperthermophilic Archaeon, Pyrococcus furiosus, by a novel method effectively using its cosmid protein library, sequenced and expressed in Escherichia coli. The DNA sequence encodes a protein containing 295 amino acid residues with methionine at the N-terminus. From protein analyses of the recombinant protein expressed in E. coli, by using both amino acid sequence analysis from the N-terminus by automated Edman degradation and analyses of molecular masses of the peptides generated by two enzymatic cleavages performed independently, digestions with lysylendopeptidase and Endoproteinase Asp-N, with ionspray mass spectrometry, the primary structure of the protein has been elucidated to be completely identical with that deduced from its DNA sequence. Comparison of the amino acid sequence of P. furiosus MAP (P.f. MAP) with those of other MAPs from Eukarya and Bacteria showed that the protein has a high degree of sequence homology in the stretches surrounding the five cobalt-binding residues fully preserved in all of MAPs determined so far, but P.f. MAP belongs to Type II because it has an extra long insertion of about 60 amino acid residues between the fourth and fifth cobalt-binding ligands, similar to MAPs from human and rat, and to Met-AP2 from Saccharomyces cerevisiae, in comparison to Type I MAPs from Bacteria. Therefore, P.f. MAP seems to be rather close to those from Eukarya, although it is distinct in lacking the N-terminal extension of about 90-150 residues universally found in MAPs from Eukarya. These findings suggest that P.f. MAP is evolutionally located at the Eukarya-Bacteria boundary. The enzyme expressed in E. coli exhibits a considerable thermostability, with a half-life of approximately 4.5 h at 90 degrees C and an optimum temperature of around 90 degrees C.

Molecular Cloning, Sequencing, and Heterologous Expression of a Novel Zinc-Containing Ferredoxin Gene from a Thermoacidophilic ArchaeonSulfolobusSp. Strain 7

The gene encoding a novel zinc-containing ferredoxin from a hyperthermophilic and acidophilic archaeon (archaebacterium)Sulfolobussp. strain 7 was cloned and sequenced. The DNA sequence predicts a 103 residue protein after removal of N-terminal methionine, which is in good agreement with the results of the protein analysis. Surprisingly, the residues responsible for binding a zinc atom were conserved among three other thermoacidophilic archaea. A common sequence stretch VXGXHXGHX8-17PXXLGXHGTX38-56KXDPV is proposed as a new zinc-binding motif, where three histidines and an aspartic acid are ligated to a zinc atom. The ferredoxin gene was expressed inEschericia coli.The recombinant ferredoxin was indistinguishable from the protein purified fromSulfolobussp. strain 7 cells by several criteria so far investigated except that the methylation of the 29th lysine was suppressed.

Protein NH2-terminal asparagine deamidase. Isolation and characterization of a new enzyme.

An apparently unique enzyme, designated protein NH2-terminal asparagine deamidase (PNAD), that specifically converts NH2-terminal asparagine residues of peptide and protein substrates to aspartic acid, has been isolated to homogeneity from porcine liver by an eight-step procedure. PNAD is a relatively low abundance protein, is readily solubilized, and exists as a monomeric species of approximately 33 kDa. PNAD does not act on internal asparagine residues and requires a free N alpha-amino group. It has reduced or no activity on NH2-terminal asparagine dipeptides and no activity toward free asparagine or asparagine amide. It does not act on any NH2-terminal glutamine substrates. PNAD does not show a strong pH dependence suggesting that the enzyme can act equally well on substrates with ionized or unionized alpha-amino groups. The properties and specificity of PNAD are consistent with those expected for the enzyme required for the ubiquitin-dependent turnover of intracellular proteins that initiate with Met-Asn-. Such proteins should be N alpha-acetylated on the retained initiator methionine and can subsequently be modified by the removal of N-acetyl methionine by acylaminoacid hydrolase. Conversion of the resulting NH2-terminal asparagine to aspartic acid by PNAD would render the protein susceptible to arginylation, polyubiquitinylation and degradation as specified by the N-end rule.

Balanced amino acid composition essential for infusion-induced egg development in the mosquito ( Culex pipiens pallens)

Amino acid mixtures, in which one or more amino acids were omitted from a basal mixture of 17 amino acids, were infused into the hemocoels of female Culex pipiens pallens mosquitoes. The resulting oögenesis was compared with previous studies of oögenesis induced by the basal mixture to evaluate the oögenic stimulatory effect of individual amino acids. Infusion of solutions deficient in isoleucine, leucine, lysine, phenylalanine, threonine, tryptophan or valine, which are known to be absolutely essential for producing mature eggs, failed even to initiate oögenesis. Removal of arginine (also absolutely essential for egg production), cysteine, histidine or methionine from the basal mixture resulted in decreased activation of oögenesis. Single elimination of other amino acids (alanine, glycine, proline and serine) resulted in high percentages of oögenesis and egg maturation, comparable to the levels produced by basal mixture infusion. The combination of asparagine and glutamine, which were reported to increase in the hemolymph of blood-fed females, can be substituted for asparagine and glutamate in the basal mixture to give a high level of oögenesis. On the other hand, infusion of a mixture comprised of the eight amino acids absolutely essential for egg production showed only a slight stimulation of oögenesis initiation. Single amino acid infusion of isoleucine or threonine also failed to stimulate mosquito oögenesis. Thus, we have concluded that initiation of mosquito oögenesis requires an appropriate balance in increased hemolymph amino acids rather than just one or a few special trigger amino acids.

HPr polymorphism in oral streptococci is caused by the partial removal of the N-terminal methionine

HPr is a small phosphocarrier protein of the bacterial phosphoenolpyruvate:sugar phosphotransferase system involved in the transport and phosphorylation of sugars. It has recently been reported that streptococci possess two forms of HPr having identical biochemical properties. In this communication, we show by N-terminal amino-acid sequencing and by ion-spray mass spectroscopy that these two forms differ by the presence or the absence of the N-terminal methionine.

Purification and characterization of a methionine aminopeptidase from Saccharomyces cerevisiae.

Methionine aminopeptidase (MAP), which catalyzes the removal of NH2-terminal methionine from proteins, was isolated from Saccharomyces cerevisiae. The enzyme was purified 472-fold to apparent homogeneity. The Mr of the native enzyme was estimated to be 36,000 +/- 5,000 by gel filtration chromatography, and the Mr of the denatured protein was estimated to be 34,000 +/- 2,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme has a pH optimum near 7.0, and its pI is 7.8 as determined by chromatofocusing on Mono P. The enzyme was inactivated by metalloprotease inhibitors (EDTA, o-phenanthroline and nitrilotriacetic acid), sulfhydryl-modifying reagents (HgCl2 and p-hydroxymercuribenzoic acid), and Zn2+. Yeast MAP failed to cleave methionine p-nitroanilide. Among 11 Xaa-Ala-Ser analogues (Xaa = Ala, Asp, Gln, Glu, Ile, Leu, Lys, Met, Phe, Pro, and Ser), MAP cleaved only Met-Ala-Ser. MAP also cleaved methionine from other tripeptides whose penultimate amino acid residue is relatively small and/or uncharged (e.g. Pro, Gly, Val, Thr, or Ser) but not when bulky and/or charged (Arg. His, Leu, Met, or Tyr). Yeast MAP displayed similar substrate specificities compared with those of Escherichia coli (Ben-Bassat, A., Bauer, K., Chang, S.Y., Myambo, K., Boosman, A., and Chang, S. (1987) J. Bacteriol. 169, 751-757) and Salmonella typhimurium MAP (Miller, C., Strauch, K. L., Kukral, A. M., Miller, J. L., Wingfield, P. T., Mazzei, G. J., Werlen, R. C., Garber, P., and Movva, N. R. (1987) Proc. Natl, Acad. Sci. U.S.A. 84, 2718-2722). In general, the in vitro specificity of yeast MAP is consistent with the specificity observed in previous in vivo studies in yeast (reviewed in Arfin, S. M., and Bradshaw, R. A. (1988) Biochemistry 27, 7979-7984).

In vivo processing of N-terminal methionine in E. coli

The processing of amino-terminal methionine from cytosolic proteins in E. coli has been investigated in vivo, using amino-terminal-extended human growth hormone (hGH) as a model system. Twenty different hGH-genes with the sequence Met-Xxx-Glu-Glu-hGH where Xxx denotes each of the 20 different amino acids, were constructed and expressed in E. coli. Following purification of the products, the N-terminal amino acid sequences (10 cycles) were determined. The results demonstrate that the removal of methionine is dependent on the amino acid adjacent to methionine, and that the processing is strongly correlated to the radius of gyration of this amino acid. In addition, measurement of the hGH expression level from the 20 clones demonstrated that the small difference in the amino acid extension leads to a change in the specific hGH expression rate.

Inhibition of GSH efflux from rat liver by methionine: effects of GSH synthesis in cells and perfused organ

The inhibition of efflux of intracellular reduced glutathione (GSH) by methionine was determined in isolated rat hepatocytes suspended either in Krebs-Henseleit buffer or in modified Fisher's medium. Methionine (1 mM) added to Krebs-Henseleit suspensions of isolated rat hepatocytes inhibited GSH efflux, with greater retention of GSH in the cells compared with control. Results were similar with methionine and 0.3 mM propargylglycine cystathionase inhibitor), suggesting no net synthesis of GSH from methionine. In Fisher's medium, the inhibitory effect of methionine on GSH efflux was masked due to increasing cellular GSH; however, the inhibitory effect of methionine was unmasked by propargylglycine, which prevented the utilization of methionine for GSH synthesis. The addition of serine (0.1 mM) to methionine in Krebs-Henseleit buffer raised cellular GSH, overcoming the inhibition of GSH efflux. In the perfused liver, infusion of 1 and 5 mM methionine initially inhibited GSH efflux, but the inhibition was reversed with continued methionine infusion. After removal of methionine, GSH efflux increased immediately. The reversal and rebound were blocked by propargylglycine, revealing concentration-dependent inhibition of sinusoidal GSH efflux by methionine. Thus, when methionine is utilized to promote GSH synthesis, its inhibitory effect on GSH efflux tends to be overcome.

RAS2 protein of Saccharomyces cerevisiae undergoes removal of methionine at N terminus and removal of three amino acids at C terminus.

RAS2 protein of Saccharomyces cerevisiae undergoes post-translational modifications involving methyl esterification and palmitic acid addition, resulting in their association with the plasma membrane. In this paper, we provide evidence that two kinds of proteolytic events accompany the biosynthesis. This is shown by separating and characterizing three intracellular forms of RAS2 protein: precursor, intermediate, and mature (fatty acid-acylated) forms. N-Terminal sequencing has revealed that all three forms start with proline, which is the second amino acid expected from the RAS2 gene sequence. Thus, the first methionine is removed very early during the biosynthesis. Isolation and sequencing of C-terminal peptides indicate that three C-terminal amino acids present in the precursor form are removed in the intermediate and in the fatty acid acylated forms. C-Terminal proteolysis appears to accompany methyl esterification, since the methylation occurs with the intermediate and the fatty acid-acylated forms, but not with the precursor. Palmitic acid is identified as the major fatty acid attached to the fatty acid-acylated form.

Specificity of cotranslational amino-terminal processing of proteins in yeast.

Polypeptides synthesized in the cytoplasm of eukaryotes are generally initiated with methionine, but N-terminal methionine is absent from most mature proteins. Many proteins are also N alpha-acetylated. The removal of N-terminal methionine and N alpha-acetylation are catalyzed by two enzymes during translation. The substrate preferences of the methionine aminopeptidase (EC 3.4.11.x) and N alpha-acetyltransferase (EC 2.3.1.x) have been partially inferred from the distribution of amino-terminal residues and/or mutations found for appropriate mature proteins, but with some contradictions. In this study, a synthetic gene corresponding to the mature amino acid sequence of the plant protein thaumatin, expressed in yeast as a nonexported protein, i.e., lacking a signal peptide, has been used to delineate the specificities of these enzymes with respect to the penultimate amino acid. Site-directed mutagenesis, employing synthetic oligonucleotides, was utilized to construct genes encoding each of the 20 amino acids following the initiation methionine codon, and each protein derivative was isolated and characterized with respect to its amino-terminal structure. All four possible N-terminal variants--those with and without methionine and those with and without N alpha-acetylation--were obtained. These results define the specificity of these enzymes in situ and suggest that the nature of the penultimate amino-terminal residue is the major determinant of their selectivity.

The state of the N-terminus of recombinant proteins: Determination of N-terminal methionine (formylated, acetylated, or free)

The removal of N-terminal methionine from proteins produced by recombinant DNA techniques is often far from quantitative. Furthermore, a proportion of the methionylated product may be N α-blocked and thus not easily accessible to conventional (Edman) techniques of protein characterization. In this paper, a method for overcoming the resulting analytical problems is described. The technique is based on perdeuteroacetylation (performed only if unblocked methionine is to be determined), cleavage with cyanogen bromide, extraction of any acylhomoserine lactone into ethyl acetate, formation of a chemical derivative, and analysis by combined gas-liquid chromatography/mass spectrometry (GC/MS). The remaining cyanogen bromide fragments, insoluble in ethyl acetate, are available for further analysis by mass spectrometric or other methods if required. Using an acylhomoserine lactone labeled with a stable isotope as internal standard, the method is semiquantitative. It should be possible to develop a quantitative method if appropriate polypeptide standards are prepared. N-Terminal processing of eight recombinant-derived proteins is discussed.

N-terminal methionine-specific peptidase in Salmonella typhimurium.

Crude extracts of a multiply peptidase-deficient strain of Salmonella typhimurium contain an aminopeptidase that specifically removes N-terminal methionine from peptides. This activity shows pronounced specificity for the peptide's second amino acid. Methionine is removed from peptides with alanine, threonine, or glycine in this position but not when the second amino acid is leucine or methionine. The activity is stimulated by Co2+ and is inhibited by EDTA. Mutations that lead to overproduction (up to 30-fold) of the activity have been obtained by selecting for growth on Met-Gly-Gly as a methionine source. These mutations map at approximately 3 map units, phage P22 cotransducible with leu. The overproducer mutations are dominant to wild type, and duplication of the wild-type allele of the locus leads to a gene dosage effect on peptidase levels. This suggests that the locus of the overproducer mutations may be the structural gene for the peptidase. NaDodSO4/PAGE shows an increased level of a single protein (34 kDa) in the overproducer mutant. This protein is highly enriched in a purified preparation of the peptidase. The specificity of this enzyme suggests that it is involved in the cleavage of methionine from newly synthesized peptide chains. This activity can specifically remove methionine from the N terminus of a completed protein. Treatment of purified, unprocessed (N-terminal methionine) interleukin 1 beta with the purified peptidase results in removal of N-terminal methionine with no additional alterations. N-terminal processing of at least this protein can occur after translation is complete. We propose to call this enzyme peptidase M (methionine-specific aminopeptidase).

Processing of the initiation methionine from proteins: properties of the Escherichia coli methionine aminopeptidase and its gene structure.

Methionine aminopeptidase (MAP) catalyzes the removal of amino-terminal methionine from proteins. The Escherichia coli map gene encoding this enzyme was cloned; it consists of 264 codons and encodes a monomeric enzyme of 29,333 daltons. In vitro analyses with purified enzyme indicated that MAP is a metallo-oligopeptidase with absolute specificity for the amino-terminal methionine. The methionine residues from the amino-terminal end of the recombinant proteins interleukin-2 (Met-Ala-Pro-IL-2) and ricin A (Met-Ile-Phe-ricin A) could be removed either in vitro with purified MAP enzyme or in vivo in MAP-hyperproducing strains of E. coli. In vitro analyses of the substrate preference of the E. coli MAP indicated that the residues adjacent to the initiation methionine could significantly influence the methionine cleavage process. This conclusion is consistent, in general, with the deduced specificity of the enzyme based on the analysis of known amino-terminal sequences of intracellular proteins (S. Tsunasawa, J. W. Stewart, and F. Sherman, J. Biol. Chem. 260:5382-5391, 1985).

Sequence determinants of cytosolic N-terminal protein processing.

N-terminal methionine removal has been analyzed statistically in a large sample of prokaryotic and eukaryotic cytosolic proteins in an attempt to uncover common sequence determinants. We find that the residue next to the initiator Met is the most important determinant of N-terminal processing: Lys, Arg, Leu and (in prokaryotes) Phe and Ile protect the initiator Met from being removed when next to it in the sequence; Ala, Gly, Pro, Ser, Thr and (in eukaryotes) Val in this position cause its removal. Subsequent acetylation is confirmed to be strongly biased towards Ala, Met and Ser residues; when Met is acetylated, Asp is the predominant penultimate residue in eukaryotes. Also, we find major differences in the relative abundance of the various residues next to the initiator Met between prokaryotes and eukaryotes: prokaryotic proteins are much more biased towards Lys as the Met-protecting residue, and towards Ala when met is to be removed, than eukaryotic ones. Finally, we show that our results can explain a part of the mRNA 'consensus sequence' found around eukaryotic initiator AUG codons.

Methionine-sensitive glycolysis in transformed cells.

Glycolysis in several tumor cell lines grown in tissue culture was inhibited by methionine. Kirsten murine sarcoma virus-transformed rat kidney cells (K-NRK) were inhibited 60-75% by 10 mM methionine, whereas normal rat kidney (NRK-49F) cells showed little or no inhibition. Inhibition of glycolysis in K-NRK cells was manifest 2-4 hr after exposure to the amino acid. Glycolysis in a chemically transformed cell line of Madin-Darby canine kidney cells was also sensitive to methionine, but maximal inhibition (75%) required 18-24 hr of incubation with the amino acid. Under the same conditions glycolysis in the nontransformed canine cells was less than 20% inhibited by methionine. In Ehrlich ascites tumor cells grown in tissue culture, 10 mM methionine inhibited glycolysis by about 50%. Inhibition of glycolysis, even by 50 mM methionine, was rapidly reversible. Within 2 hr after removal of methionine the rate of glycolytic activity was restored to that observed in control cells. Furthermore, inhibition by methionine required a minimum level (7%) of serum in the growth medium and inhibition was not sensitive to cycloheximide. Only amino acids that are transported by system A (including the nonmetabolized analogue methylaminoisobutyric acid) specifically inhibited glycolysis in tumor cells. The only exception was phenylalanine, which was toxic to both transformed and normal cell lines.

Purification of biotinidase from human plasma and its activity on biotinyl peptides.

Biotinidase catalyzes the hydrolysis of N epsilon-biotinyllysine (biocytin) to form biotin and free lysine. The enzyme has been purified 4800-fold from outdated human plasma and was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to have a molecular weight of (76 +/- 2) X 10(3). The same molecular weight was found by molecular sieve chromatography under nondenaturing conditions, indicating biotinidase is a monomer. This value is in contrast to a molecular weight of 115 000 determined by Pispa [Pispa, J. (1965) Ann. Med. Exp. Biol. Fenn., Suppl. 5, 5-39] with an impure biotinidase. The Km for biocytin was 6.2 X 10(-6) M, and biotinidase was found to be sensitive to phenylmethanesulfonamide and iodoacetamide in agreement with earlier studies by Knappe and co-workers [Knappe, J., Brümmer, W., & Bierderbick, K. (1963) Biochem. Z. 338, 599-613], who suggested that serine hydroxyl groups and sulfhydryl groups are essential for enzymatic activity. The specificity of biotinidase was examined by using synthetic and natural biotinyl peptides isolated by specific proteolytic cleavage of the biotinyl subunit of transcarboxylase. It was found that the rate of hydrolysis of biocytin was 83-fold higher than that found for biotin-containing peptides 5-13 residues in length. Removal of methionine from either side of the conserved region around the biocytin did not greatly alter the rate of cleavage. Increasing the peptide to 65-123 residues in length decreased the rate 1200-fold compared to that of biocytin.(ABSTRACT TRUNCATED AT 250 WORDS)

Adaptive regulatory control of System A transport activity in a kidney epithelial cell line (MDCK) and in a transformed variant (MDCK-T1).

Adaptive regulatory control of System A activity was investigated using MDCK cells and a chemically induced, oncogenic transformant of MDCK cells, MDCK-T1. Within 7 hours after transfer to an amino-acid-deficient medium, A activity of subconfluent MDCK cells had maximally derepressed, but this activity in confluent MDCK cells and in subconfluent transformed cells showed little capacity for derepression. Amino-acid-starved, subconfluent MDCK cells were used to study trans-inhibition and repression of A activity by individual amino acids. Trans-inhibition and repression were defined as the cycloheximide-insensitive and cycloheximide-sensitive components, respectively, of the total inhibition. Trans-inhibition correlated well with substrate affinity, but repression did not. Trans-inhibition and repression were further characterized using alpha-(methylamino) isobutyric acid (mAIB), a trans-inhibitor, and glutamate, an effective repressor. The apparent initial T 1/2 for inhibition by mAIB in the presence of cycloheximide was 0.5 hours, while that for repression by glutamate was 4.7 hours. Half-maximal inhibition by mAIB and repression by glutamate occurred at approximately 0.02 mM and 0.07 mM, respectively. Reversal of trans-inhibition by methionine occurred in the presence of cycloheximide within 1-4 hours after removal of methionine. The A system of the transformed MDCK-T1 cells showed elevated activity, little capacity for derepression, resistance to repression by amino acids, but retention of sensitivity to trans-inhibition. Kinetic analysis of mAIB uptake indicated that the A system of MDCK-T1 cells has become kinetically more complex in a manner which resembled amino-acid-starved rather than amino-acid-fed MDCK cells. These results suggest that the A system of MDCK-T1 cells has become resistant to adaptive regulatory control.

Microsomal methionine aminopeptidase: Properties of the detergent-solubilized enzyme

1. 1. A methionine aminopeptidase (MAP) found in rat liver microsomes behaves as membranebound enzyme. 2. 2. Triton-solubilized MAP when chromatographed on DEAE-cellulose columns was separated from other microsomal arylamidases. 3. 3. The enzyme hydrolyzes N-terminal methionine from methionyl-lysyl-bradykinin (Met-Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) being then characterized as a typical aminopeptidase. It also shows preferential arylamidase activity upon Met-2-naphthylamide. 4. 4. MAP was activated by 2-mercaptoethanol and inhibited by p-hydroxymercuribenzoate. 5. 5. Contrarily to other well characterized aminopeptidases, MAP was not affected by EDTA. puromycin or bestatin. 6. 6. Altogether these data suggest that MAP is a unique microsomal enzyme distinct from other previously described aminopeptidases. It could be involved in the removal of methionine from nascent peptides during protein synthesis.

Transcellular ion currents in the water mold Achlya. Amino acid proton symport as a mechanism of current entry.

Achlya, like other tip-growing organisms, generates an endogenous electrical current such that positive charge flows into the hyphal apex and exits from the trunk. The present study is concerned with the mechanism of current generation by hyphae growing in a defined, complete medium. The intensity of the current, measured in the extracellular medium with a vibrating probe, was unaffected by the removal of all the inorganic constituents of the growth medium. However, an increase in the external pH or the deletion of amino acids abolished the current. Removal of methionine alone diminished the current by two thirds. Hyphae also generated a longitudinal pH gradient in the extracellular medium; the region surrounding the tip was more alkaline than the bulk medium, whereas the region around the trunk was relatively acidic. These findings suggest that a flux of protons, dependent upon amino acids in the medium, carries current into the tip and creates the surrounding alkaline zone. The proton current appears to result from the transport of amino acids rather than their metabolism. Conditions that abolished the current also inhibited methionine uptake but had little effect on the respiratory rate. The findings imply a connection between the proton current and chemiosmotic energy transduction. We propose that protons flow into the hyphal tip through amino acid/proton symporters that are preferentially localized there. The proton flux energizes the uptake of amino acids into the growing zone and may also contribute to the polarization of hyphal growth.