A determinant in the cytoplasmic tail of the cation-dependent mannose 6-phosphate receptor prevents trafficking to lysosomes

The bovine cation-dependent mannose 6-phosphate receptor (CD-MPR) is a type 1 transmembrane protein that cycles between the trans-Golgi network, endosomes, and the plasma membrane. When the terminal 40 residues were deleted from the 67-amino acid cytoplasmic tail of the CD-MPR, the half-life of the receptor was drastically decreased and the mutant receptor was recovered in lysosomes. Analysis of additional cytoplasmic tail truncation mutants and alanine-scanning mutants implicated amino acids 34-39 as being critical for avoidance of lysosomal degradation. The cytoplasmic tail of the CD-MPR was partially effective in preventing the lysosomal membrane protein Lamp1 from entering lysosomes. Complete exclusion required both the CD-MPR cytoplasmic tail and transmembrane domain. The transmembrane domain alone had just a minor effect on the distribution of Lamp1. These findings indicate that the cytoplasmic tail of the CD-MPR contains a signal that prevents the receptor from trafficking to lysosomes. The transmembrane domain of the CD-MPR also contributes to this function.     

A leucine-based determinant in the epidermal growth factor receptor juxtamembrane domain is required for the efficient transport of ligand-receptor complexes to lysosomes

Ligand binding causes the epidermal growth factor (EGF) receptor to undergo accelerated internalization with eventual degradation in lysosomes. The goal of this study was to investigate the molecular basis of endocytic sorting, focussing on post-internalization events. We have identified a sequence located between amino acid residues 675 and 697, encompassing a dileucine motif at residues 679 and 680, that enhances endosome-to-lysosome transport when conformational restraints in the EGF receptor carboxyl terminus are removed by truncation. The same dileucine motif is also necessary for efficient lysosomal transport of ligand-occupied full-length EGF receptors. A L679A,L680A substitution diminished the degradation of occupied full-length EGF receptors without affecting internalization but had a significant effect on recycling. Rapid recycling of mutant receptors resulted in reduced intracellular retention of occupied EGF receptors and delayed down-regulation of cell surface receptors. We propose that the L679A,L680A substitution acts primarily to impair transport of ligand-receptor complexes through an early endosomal compartment, diverting occupied receptors to a recycling compartment at the expense of incorporation into lysosome transport vesicles. We also found that mutant receptors with truncations at the distal half of tyrosine kinase domain (residues 809-957) were not efficiently delivered to the cell surface but were destroyed in an endoplasmic reticulum-associated degradative pathway.     

Tryptophan 388 in putative transmembrane segment 10 of the rat glucose transporter Glut1 is essential for glucose transport

The molecular mechanism of substrate recognition in membrane transport is not well understood. Two amino acid residues, Tyr446 and Trp455 in transmembrane segment 10 (TM10), have been shown to be important for galactose recognition by the yeast Gal2 transporter; Tyr446 was found to be essential in that its replacement by any of the other 19 amino acids abolished transport activity (Kasahara, M., Shimoda, E., and Maeda, M. (1997) J. Biol. Chem. 272, 16721-16724). The Glut1 glucose transporter of animal cells belongs to the same Glut transporter family as does Gal2 and thus might be expected to show a similar mechanism of substrate recognition. The role of the two amino acids, Phe379 and Trp388, in rat Glut1 corresponding to Tyr446 and Trp455 of Gal2 was therefore studied. Phe379 and Trp388 were individually replaced with each of the other 19 amino acids, and the mutant Glut1 transporters were expressed in yeast. The expression level of most mutants was similar to that of the wild-type Glut1, as revealed by immunoblot analysis. Glucose transport activity was assessed by reconstituting a crude membrane fraction of the yeast cells in liposomes. No significant glucose transport activity was observed with any of Trp388 mutants, whereas the Phe379 mutants showed reduced or no activity. These results indicate that the two aromatic amino acids in TM10 of Glut1 are important for glucose transport. However, unlike Gal2, the residue at the cytoplasmic end of TM10 (Trp388, corresponding to Trp455 of Gal2), rather than that in the middle of TM10 (Phe379, corresponding to Tyr446 of Gal2), is essential for transport activity.     

Expression of inducible nitric oxide synthase and inflammatory cytokines in alveolar macrophages of ARDS following sepsis

Cytochrome bo is a four-subunit quinol oxidase in the aerobic respiratory chain of Escherichia coli and functions as a redox-coupled proton pump. Subunit I binds all the redox metal centers, low-spin heme b, high-spin heme o, and CuB, whose axial ligands have been identified to be six invariant histidines. This work explored the possible roles of the aromatic amino acid residues conserved in the putative transmembrane helices (or at the boundary of the membrane) of subunit I. Sixteen aromatic amino acid residues were individually substituted by Leu, except for Tyr61 and Trp282 by Phe and Phe415 by Trp. Leu substitutions of Trp280 and Tyr288 in helix VI, Trp331 in loop VII-VIII, and Phe348 in helix VIII reduced the catalytic activity, whereas all other mutations did not affect the in vivo activity. Spectroscopic analyses of the purified mutant enzymes revealed that the defects were attributable to perturbations of the binuclear center. On the basis of these findings and recent crystallographic studies on cytochrome c oxidases, we discuss the possible roles of the conserved aromatic amino acid residues in subunit I of the heme-copper terminal oxidases.     

A balance of opposing signals within the cytoplasmic tail controls the lysosomal targeting of P-selectin

The 35-amino acid cytoplasmic tail of the adhesion receptor P-selectin is subdivided into stop transfer, C1 and C2 domains. It contains structural signals needed for targeting this protein to specialized secretory organelles and to lysosomes. Recently, using site-directed mutagenesis of horseradish peroxidase-P-selectin chimeras, we have uncovered a novel sequence within the C1 domain, KCPL, that mediates sorting from early, transferrin-positive endosomes to lysosomes and therefore operates as a positive lysosomal targeting signal (Blagoveshchenskaya, A. D., Norcott, J. P. , and Cutler, D. F. (1998) J. Biol. Chem. 273, 2729-2737). In the current study, we examined lysosomal targeting by both subcellular fractionation and an intracellular proteolysis assay and found that a balance of positive and negative signals is required for proper lysosomal sorting of P-selectin. First, we have found that within the sequence KCPL, Cys-766 plays a major role along with Pro-767, whereas Lys-765 and Leu-768 make no contribution to promoting lysosomal targeting. In addition, horseradish peroxidase-P-selectin chimeras were capable of acylation in vivo with [3H]palmitic acid at Cys-766, since no labeling of a chimera in which Cys-766 was replaced with Ala was detected. Second, analysis of mutations within the C2 domain revealed that substitution of two sequences, YGVF and DPSP, causes an increase in both lysosomal targeting and intracellular proteolysis suggesting the presence of lysosomal avoidance signals. The inhibition or promotion of lysosomal targeting resulted from alterations in endosomal sorting since internalization was not changed in parallel with lysosomal delivery. Analysis of the double mutants KCPL/YGVF or KCPL/DPSP revealed that although the positive lysosomal targeting signal operates in the early/sorting transferrin-positive endosomes, the negative lysosomal targeting (lysosomal avoidance) signals act at later stages of the endocytic pathway, most likely in late endosomal compartments.     

Functional roles of the conserved aromatic amino acid residues at position 108 (motif IV) and position 196 (motif VIII) in base flipping and catalysis by the N6-adenine DNA methyltransferase from Thermus aquaticus

The DNA methyltransferase (Mtase) from Thermus aquaticus (M.TaqI) catalyzes the transfer of the activated methyl group of S-adenosyl-L-methionine to the N6 position of adenine within the double-stranded DNA sequence 5'-TCGA-3'. To achieve catalysis M.TaqI flips the target adenine out of the DNA helix. On the basis of the three-dimensional structure of M.TaqI in complex with the cofactor and its structural homology to the C5-cytosine DNA Mtase from Haemophilus haemolyticus, Tyr 108 and Phe 196 were suggested to interact with the extrahelical adenine. The functional roles of these two aromatic amino acid residues in M.TaqI were investigated by mutational analysis. The obtained mutant Mtases were analyzed in an improved kinetic assay, and their ability to flip the target base was studied in a fluorescence-based assay using a duplex oligodeoxynucleotide containing the fluorescent base analogue 2-aminopurine at the target position. While the mutant Mtases containing the aromatic amino acid Trp at position 108 or 196 (Y108W and F196W) showed almost wild-type catalytic activity, the mutant Mtases with the nonaromatic amino acid Ala (Y108A and F196A) had a strongly reduced catalytic constant. Y108A was still able to flip the target base, whereas F196A was strongly impaired in base flipping. These results indicate that Phe 196 is important for stabilizing the extrahelical target adenine and suggest that Tyr 108 is involved in placing the extrahelical target base in an optimal position for methyl group transfer. Since both aromatic amino acids belong to the conserved motifs IV and XIII found in N6-adenine and N4-cytosine DNA Mtases as well as in N6-adenine RNA Mtases, a similar function of aromatic amino acid residues within these motifs is expected for the different Mtases.     

Extensive random mutagenesis analysis of the Na+/K+-ATPase alpha subunit identifies known and previously unidentified amino acid residues that alter ouabain sensitivity--implications for ouabain binding

Random mutagenesis with ouabain selection has been used to comprehensively scan the extracellular and transmembrane domains of the alpha1 subunit of the sheep Na+/K+-ATPase for amino acid residues that alter ouabain sensitivity. The four random mutant libraries used in this study include all of the transmembrane and extracellular regions of the molecule as well as 75% of the cytoplasmic domains. Through an extensive number of HeLa cell transfections of these libraries and subsequent ouabain selection, 24 ouabain-resistant clones have been identified. All previously described amino acids that confer ouabain resistance were identified, confirming the completeness of this random mutagenesis screen. The amino acid substitutions that confer the greatest ouabain resistance, such as Gln111-->Arg, Asp121-->Gly, Asp121-->Glu, Asn122-->Asp, and Thr797-->Ala were identified more than once in this study. This extensive survey of the extracellular and transmembrane regions of the Na+/K+-ATPase molecule has identified two new regions of the molecule that affect ouabain sensitivity: the H4 and the H10 transmembrane regions. The new substitutions identified in this study are Leu330-->Gln, Ala331-->Gly, Thr338-->Ala, and Thr338-->Asn in the H4 transmembrane domain and Phe982-->Ser in the H10 transmembrane domain. These substitutions confer modest increases in the concentration of cardiac glycoside needed to produce 50% inhibition of activity (IC50 values), 3.1-7.9-fold difference. The results of this extensive screening of the Na+/K+-ATPase alpha1 subunit to identify amino acids residues that are important in ouabain sensitivity further supports our hypothesis that the H1-H2 and H4-H8 regions represent the major binding sites for the cardiac glycoside class of drugs.     

Environment- and sequence-dependent modulation of the double-stranded to single-stranded conformational transition of gramicidin A in membranes

The role of the membrane lipid composition and the individual Trp residues in the conformational rearrangement of gramicidin A along the folding pathway to its channel conformation has been examined in phospholipid bilayers by means of previously described size-exclusion high-performance liquid chromatography HPLC-based strategy (Ba?ó et al. (1991) Biochemistry 30, 886). It has been demonstrated that the chemical composition of the membrane influences the transition rate of the peptide rearrangement from double-stranded dimers to beta-helical monomers. The chemical modification of Trp residues, or its substitution by the more hydrophobic residues phenylalanine or naphthylalanine, stabilized the double-stranded dimer conformation in model membranes. This effect was more notable as the number of Trp-substituted residues increased (tetra > tri > di > mono), and it was also influenced by the specific position of the substituted amino acid residue in the sequence, in the order Trp-9 approximately Trp-13 > Trp-11 > Trp-15. Moreover, it was verified that nearly a full contingent of indoles (Trp-13, -11, and -9) is necessary to induce a quantitative conversion from double-stranded dimers to single-stranded monomers, although Trp-9 and Trp-13 seemed to be key residues for the stabilization of the beta-helical monomeric conformation of gramicidin A. The conformation adopted for monomeric Trp --> Phe substitution analogues in lipid vesicles resulted in CD spectra similar to the typical single-stranded beta6.3-helical conformation of gramicidin A. However, the Trp --> Phe substitution analogues showed decreased antibiotic activity as the number of Trp decreased.     

The Environmental Rating Scale (ERS): a measure of the quality of the residential environment for adults with autism

In the native state of proteins there is a marked tendency for an aromatic amino acid to precede a cis proline. There are also significant differences between the three aromatic amino acids with Tyr exhibiting a noticeably higher propensity than Phe or Trp to precede a cis proline residue. In order to study the role that local interactions play in these conformation preferences, a set of tetrapeptides of the general sequence acetyl-Gly-X-Pro-Gly-carboxamide (GXPG), where X = Tyr, Phe, Trp, Ala, or cyclohexyl alanine, were synthesized and studied by nmr. Analysis of the nmr data shows that none of the peptides adopt a specific backbone structure. Ring current shifts, the equilibrium constant, the Van't Hoff enthalpy, and the measured rate of cis-trans isomerization all indicate that the cis proline conformer is stabilized by favorable interactions between the aromatic ring and the proline residue. Analysis of the side chain conformation of the aromatic residue and analysis of the chemical shifts of the pyrrolidine ring protons shows that the aromatic side chain adopts a preferred conformation in the cis form. The distribution of rotamers and the effect of an aromatic residue on the cis-trans equilibrium indicate that the preferred conformation is populated to approximately 62% for the Phe containing peptide, 67% for the Tyr containing peptide, and between 75 and 80% for the Trp containing peptide. The interaction is unaffected by the addition of 8M urea. These local interactions favor an aromatic residue immediately preceding a cis proline, but they cannot explain the relative propensities for Phe-Pro, Tyr-Pro, and Trp-Pro cis peptide bonds observed in the native state of proteins. In the model peptides the percentage of the cis proline conformer is 21% GYPG while it is 17% for GFPG. This difference is considerably smaller than the almost three to one preponderance observed for cis Tyr-Pro peptide bonds vs cis Phe-Pro peptide bonds in the protein database.     

Residues in the membrane-spanning and extracellular loop regions of the parathyroid hormone (PTH)-2 receptor determine signaling selectivity for PTH and PTH-related peptide

The parathyroid hormone (PTH)-2 receptor displays strong ligand selectivity in that it responds fully to PTH but not at all to PTH-related peptide (PTHrP). In contrast, the PTH-1 receptor (PTH/PTHrP receptor) responds fully to both ligands. Previously it was shown that two divergent residues in PTH and PTHrP account for PTH-2 receptor selectivity; position 23 (Trp in PTH and Phe in PTHrP) determines binding selectivity and position 5 (Ile in PTH and His in PTHrP) determines signaling selectivity. To identify sites in the PTH-2 receptor involved in discriminating between His5 and Ile5, we constructed PTH-2 receptor/PTH-1 receptor chimeras, expressed them in COS-7 cells, and tested for cAMP responsiveness to [Trp23] PTHrP-(1-36), and to the nondiscriminating peptide [Ile5, Trp23]PTHrP-(1-36) (the Phe23 --> Trp modification enabled high affinity binding of each ligand to the PTH-2 receptor). The chimeras revealed that the membrane-spanning/loop region of the receptor determined His5/Ile5 signaling selectivity. Subsequent analysis of smaller cassette substitutions and then individual point mutations led to the identification of two single residues that function as major determinants of residue 5 signaling selectivity. These residues, Ile244 at the extracellular end of transmembrane helix 3, and Tyr318 at the COOH-terminal portion of extracellular loop 2, are replaced by Leu and Ile in the PTH-1 receptor, respectively. The results thus indicate a functional interaction between two residues in the core region of the PTH-2 receptor and residue 5 of the ligand.     

C-capping and helix stability: the Pro C-capping motif

Here we have performed a statistical analysis of the protein database to find new putative local C-terminal motifs in alpha-helices. Our analysis shows that certain combinations of X-Pro pairs (Asn, Cys, His, Phe, Tyr, Trp, Ile, Val and Leu), in which residue X is the C-cap and the Pro is at position C', are more abundant than expected. In those pairs, except for the aliphatic residues, the presence of the Pro residue at C' tends to restrict the phi and psi dihedral angles of the residue at position C-cap, around -130 degrees , 70 degrees , respectively. For the aromatic residues as well as for His, the chi1 angle is around -60 degrees and the edge of the His and aromatic rings are close to the carbonyl group of the residue i - 4. In all the pairs having the above dihedral angles for residue C-cap, the main-chain amino group of Pro at C' is close to the last three main-chain carbonyls of the alpha-helix. The above structural arrangements suggests the existence of a stabilising electrostatic interaction of the residues at positions C-cap and C' with the helix macrodipole. We have denominated this putative local motif, the Pro-capping motif. To asses its importance in helix stability we have analysed by nuclear magnetic resonance (NMR) and far-UV circular dichroism (CD) a set of polyalanine-based peptides containing two of the above pairs: His-Pro and Phe-Pro, as well as the corresponding controls. In the case of the His-Pro pair we have found NMR evidence for the formation of the Pro-capping motif in aqueous solution. CD analysis shows that the presence of a Pro residue alters the C-cap properties of the preceding amino acids in the case of His and Phe makes them more favourable. The Pro-capping motif with the appropriate sequence, determines the location of the C terminus of alpha-helices and stabilises the helical conformation having Pro as the C' residue.     

Internalization of the lymphocytic surface protein CD22 is controlled by a novel membrane proximal cytoplasmic motif

CD22 is a key receptor on B-lymphocytes that modulates signaling during antigenic stimulation. We have defined a novel cytoplasmic motif in human CD22 that controls its unusually rapid turnover at the plasma membrane. Chimeric and mutated CD22alpha cDNA vectors were constructed and stably transfected in CD22-negative Jurkat T-lymphocytic cells. Two assays were employed to measure CD22alpha internalization: first, cytoplasmic uptake of radioiodinated anti-CD22 monoclonal antibody; and second, lethal targeting of a toxin, saporin, into cells via CD22 using bispecific F(ab')2 ([anti-CD22 x anti-saporin]) antibody. Results showed that CD22alpha lacking a cytoplasmic tail was not internalized and that replacement of the cytoplasmic tail of CD19 with that of CD22alpha resulted in a chimeric molecule that behaved like CD22alpha and internalized rapidly. Step-wise deletion of the cytoplasmic tail of CD22alpha located the internalization motif to a polar region of 11 residues (QRRWKRTQSQQ) proximal to the plasma membrane, a part of the molecule predicted to form a coil or turn structure. Interestingly, additional CD22 mutants showed that the two glutamine residues sandwiching the serine are critical to internalization but that the serine itself is not.     

Importance of extracellular domains for ligand binding in the thyrotropin-releasing hormone receptor

The role of putative extracellular sequences for ligand binding in the TRH receptor was examined using deletion or substitution mutations. Each mutant receptor was transiently expressed in TRH receptor-minus GH(1)2C(1)b rat pituitary cells, and binding of 4 Nu Mu [3H]pGlu-N(tau)-MeHis-Pro-NH2 ([3H] MeTRH) was measured. When binding was not detected, signal transduction at 10 microM MeTRH was measured to assess receptor expression. Deletion of most of the N-terminal sequences (Glu(2)-Leu(22)), including two potential glycosylation sites, had no effect on the affinity of the receptor for MeTRH. Segmental deletions or simultaneous substitution of multiple amino acid residues in the first, second, or third extracellular loop (EL1, EL2, or EL3) resulted, however, in total loss of [3H]MeTRH binding, suggesting important roles for the loop sequences in either receptor expression or ligand binding. Individual substitutions were made to test further the role of the specific extracellular loop sequences in TRH binding. In EL1, conversion of Tyr93 to Ala resulted in more than 20-fold decrease in affinity for MeTRH. In EL2 and the top portion of the fifth transmembrane helix, conversion of Tyr181 to Phe, Tyr188 to Ala, and Phe199 to Ala resulted in a large ( > 100-fold) decrease in affinity for MeTRH, and conversion of Tyr 188 to Phe and Phe196 to Ala caused an agonist-specific 4- to 5-fold decrease in affinity. In EL3, conversion of Asn289 to Ala and of Ser290 to Ala caused a large ( > 100-fold) decrease in affinity for MeTRH. These results suggest important roles for the extracellular loops in high affinity TRH binding and lead us to propose a model in which TRH binds to the extra-cellular domain of its receptor.     

Molecular identification of a novel candidate sorting receptor purified from human brain by receptor-associated protein affinity chromatography

Receptor-associated protein (RAP) is an endoplasmic reticulum/Golgi protein involved in the processing of receptors of the low density lipoprotein receptor family. A approximately 95-kDa membrane glycoprotein, designated gp95/sortilin, was purified from human brain extracts by RAP affinity chromatography and cloned in a human cDNA library. The gene maps to chromosome 1p and encodes an 833-amino acid type I receptor containing an N-terminal furin cleavage site immediately preceding the N terminus determined in the purified protein. Gp95/sortilin is expressed in several tissues including brain, spinal cord, and testis. Gp95/sortilin is not related to the low density lipoprotein receptor family but shows intriguing homologies to established sorting receptors: a 140-amino acid lumenal segment of sortilin representing a hitherto unrecognized type of extracellular module shows extensive homology to corresponding segments in each of the two lumenal domains of yeast Vps10p, and the extreme C terminus of the cytoplasmic tail of sortilin contains the casein kinase phosphorylation consensus site and an adjacent dileucine sorting motif that mediate assembly protein-1 binding and lysosomal sorting of the mannose-6-phosphate receptors. Expression of a chimeric receptor containing the cytoplasmic tail of gp95/sortilin demonstrates evidence that the tail conveys colocalization with the cation-independent mannose6-phosphate receptor in endosomes and the Golgi compartment.     

Platelet endothelial cell adhesion molecule-1 is phosphorylatable by c-Src, binds Src-Src homology 2 domain, and exhibits immunoreceptor tyrosine-based activation motif-like properties

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is 130-kDa member of the immunoglobulin gene superfamily that localizes to cell-cell borders of confluent endothelial cells and has been shown to play a role in the control of endothelial sheet migration and leukocyte transmigration through the endothelium. The cytoplasmic tail plays an important role in the modulation of PECAM-1 function. Mutation of tyrosine 663 or 686 in the cytoplasmic tail reduces phosphorylation and mutation of 686 is associated with a reduction in PECAM-1-mediated inhibition of cell migration (1). We have previously noted that these two tyrosine residues are surrounded by consensus sequences for Src homology 2 (SH2) domain binding (1, 2), and the experiments presented explore the potential for PECAM-1-Src and PECAM-1-SH2 domain interactions. PECAM-1 is more highly phosphorylated in endothelial cells overexpressing c-Src, and in in vitro kinase assays, c-Src can phosphorylate a glutathione S-transferase (GST)-PECAM cytoplasmic tail fusion protein. The phosphorylated fusion protein associates with the bead-bound c-Src. This association appears to be mediated by Src-SH2 domain, because PECAM-1 can be precipitated by a GST-Src-SH2 affinity matrix. The binding to the GST-Src-SH2 affinity matrix correlates directly with the level of PECAM-1 phosphorylation, because more PECAM-1 is precipitated from c-Src overexpressors and from wild-type rather than Tyr663 --> Phe and Tyr686 --> Phe mutant PECAM-1 expressors. Yet unidentified phosphoproteins can also be coimmunoprecipitated with wild-type but not mutant PECAM-1. Finally, we note the similarity of the PECAM-1 cytoplasmic domain sequence to the immunoreceptor tyrosine-based activation motif. Our data begin to delineate how tyrosines 663 and 686 may play a role in mediating PECAM-1 signal transduction.     

Cysteine-scanning mutagenesis of helix IV and the adjoining loops in the lactose permease of Escherichia coli: Glu126 and Arg144 are essential. off

Cys-scanning mutagenesis has been applied to the remaining 45 residues in lactose permease that have not been mutagenized previously (from Gln100 to Arg144 which comprise helix IV and adjoining loops). Of the 45 single-Cys mutants, 26 accumulate lactose to > 75% of the steady state observed with Cys-less permease, and 14 mutants exhibit lower but significant levels of accumulation (35-65% of Cys-less permease). Permease with Phe140-->Cys or Lys131-->Cys exhibits low activity (15-20% of Cys-less permease), while mutants Gly115-->Cys, Glu126-->Cys and Arg144-->Cys are completely unable to accumulate the dissacharide. However, Cys-less permease with Ala or Pro in place of Gly115 is highly active, and replacement of Lys131 or Phe140 with Cys in wild-type permease has a less deleterious effect on activity. In contrast, mutant Glu126-->Cys or Arg144-->Cys is inactive with respect to both uphill and downhill transport in either Cys-less or wild-type permease. Furthermore, mutants Glu126-->Ala or Gln and Arg144-->Ala or Gln are also inactive in both backgrounds, and activity is not rescued by double neutral replacements or inversion of the charged residues at these positions. Finally, a mutant with Lys in place of Arg144 accumulates lactose to about 25% of the steady state of wild-type, but at a slow rate. Replacement of Glu126 with Asp, in contrast, has relatively little effect on activity. None of the effects can be attributed to decreased expression of the mutants, as judged by immunoblot analysis. Although the activity of most of the single-Cys mutants is unaffected by N-ethylmaleimide, Cys replacement at three positions (Ala127, Val132, or Phe138) renders the permease highly sensitive to alkylation. The results indicate that the cytoplasmic loop between helices IV and V, where insertional mutagenesis has little effect on activity [McKenna, E., et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 11954-11958], contains residues that play an important role in permease activity and that a carboxyl group at position 126 and a positive charge at position 144 are absolutely required.     

Protein folding and protein evolution: common folding nucleus in different subfamilies of c-type cytochromes?

Amino acid sequences of seven subfamilies of cytochromes c (mitochondrial cytochromes c, c1; chloroplast cytochromes c6, cf; bacterial cytochromes c2, c550, c551; in total 164 sequences) have been compared. Despite extensive homology within eukaryotic subfamilies, homology between different subfamilies is very weak. Other than the three heme-binding residues (Cys13, Cys14, His18, in numeration of horse cytochrome c) there are only four positions which are conserved in all subfamilies: Gly/Ala6, Phe/Tyr10, Leu/Val/Phe94 and Tyr/Trp/Phe97. In all 17 cytochromes c with known 3D-structures, these residues form a network of conserved contacts (6-94, 6-97, 10-94, 10-97 and 94-97). Especially strong is the contact between aromatic groups in positions 10 and 97, which corresponds to 13 interatomic contacts. As residues 6, 10 and residues 94, 97 are in (i, i+4) and (i, i+3) positions in the N and C-terminal helices, respectively, the above mentioned system of conserved contacts consists mainly of contacts between one turn of N-terminal helix and one turn of C-terminal helix. The importance of the contacts between interfaces of these helices has been confirmed by the existence of these contacts in both equilibrium and kinetic molten globule-like folding intermediates, as well as by mutational evidence that these contacts are involved in tight packing between the N and C-helices. Since these four residues are not involved in heme binding and have no other apparent functional role, their conservation in highly diverged cytochromes c suggests that they are of a critical importance for protein folding. The author assumes that they are involved in a common folding nucleus of all subfamilies of c-type cytochromes.     

Lysosomal targeting of P-selectin is mediated by a novel sequence within its cytoplasmic tail

Signals controlling the intracellular targeting of many membrane proteins are present as short sequences within their cytoplasmic domains. P-selectin is a type I membrane protein receptor for leukocytes, acting during the inflammation response. Heterologous expression experiments have demonstrated that its 35-residue cytoplasmic tail contains signals for targeting to synaptic-like microvesicles, dense-cored granules, and lysosomes. We have examined the lysosomal targeting information present within the cytoplasmic tail by site-directed mutagenesis of horseradish peroxidase-P-selectin chimeras followed by transient transfection in H.Ep.2 cells. Assaying lysosomal targeting by subcellular fractionation as well as intracellular proteolysis, we have discovered a novel lysosomal targeting signal, KCPL, located within the C1 domain of the cytoplasmic tail. Alanine substitution of this tetrapeptide reduced lysosomal targeting to the level of a tailless horseradish peroxidase-P-selectin chimera, which was previously found to be deficient in both internalization and delivery to lysosomes. A proline residue within this lysosomal targeting signal makes a major contribution to the efficiency of lysosomal targeting. A diaminobenzidine density shift procedure established that chimeras with an inactivated KCPL sequence are present within transferrin-positive compartments. Such a mutant also displays an increased level of expression at the plasma membrane. Our results indicate that the sequence KCPL within the cytoplasmic tail of P-selectin is a structural element that mediates sorting from endosomes to lysosomes.     

Appearance of T cells in the bursa of Fabricius and cecal tonsils during the acute phase of infectious bursal disease virus infection in chickens

Presteady and steady-state kinetic results on the interactions of a wild-type, and the mutant glucoamylases Trp52-->Phe and Trp317-->Phe, from Aspergillus niger with maltose, maltotriose and maltotetraose have been obtained and analyzed. The results are compared with previous ones on the mutants, Trp120-->Phe and Glu180-->Gln, and with results obtained from structure energy minimization calculations based on known three-dimensional structural data. All results are in accordance with a three-step reaction model involving two steps in the substrate binding and a rate-determining catalytic step. Trp317 and Glu180 belong to different subsites, but are placed on the same flank of the active site (beta-flank). The Trp317-->Phe and the Glu180-->Gln mutants show almost identical kinetic results: weakening of the substrate binding, mainly caused by changes in the second reaction step, and practically no change of the catalytic rate. Structure energy minimization calculations show that the same loss of Arg305 and Glu180 hydrogen bonds to the substrate occurs in the Michaelis complexes of each of these mutants. These results indicate that important interactions of the active site may be better understood from a consideration of its flanks rather than of its subsites. The results further indicate differences in the substrate binding mode of maltose and of longer substrates. Trp52 and Trp120 each interact with the catalytic acid, Glu179, and are placed on the flank (alpha-flank) of the active site opposite to Trp317, Arg305 and Glu180. Also the Trp52-->Phe and Trp120-->Phe mutants show kinetic results similar to each other. The catalytic rates are strongly reduced and the substrates are bound more strongly, mainly as a result of the formation of a more stable complex in the second reaction step. All together, the substrate binding mechanism seems to involve an initial enzyme-substrate complex, in which the beta-flank plays a minor role, except for maltose binding; this is followed by a conformational change, in which hydrogen bonds to Arg305 and Glu180 of the beta-flank are established and the correct alignment on the alpha-flank of Glu179, the general acid catalyst, governed by its flexible interactions with Trp52 and Trp120, occurs.