Cluster Of Basic Amino Acids Research Articles

Phosphorylation of vitronectin on Ser362 by protein kinase C attenuates its cleavage by plasmin.

Vitronectin, found in the extracellular matrix and in circulating blood, has an important role in the control of plasminogen activation. It was shown to be the major protein substrate in human blood fluid for a protein kinase A (PKA) released from platelets upon their physiological stimulation with thrombin. Since vitronectin was shown to have only one PKA phosphorylation site, but to contain 2-3 mol covalently bound phosphate, it was reasonable to assume that other protein kinases might phosphorylate vitronectin at other sites in the protein. We have reported earlier that human serum contains at least three protein kinases, one of which was found to be cAMP independent and to phosphorylate a repertoire of plasma proteins that was very similar to that obtained upon phosphorylation of human plasma with protein kinase C (PKC). Since there are now several examples of proteins with extracellular functions that are phosphorylated by PKC, we undertook to study the phosphorylation of vitronectin by PKC. Here, we show that vitronectin is a substrate for PKC, and characterize the kinetic parameters of this phosphorylation (Km approximately tenfold lower than the concentration of vitronectin in blood), indicating that, from the biochemical point of view, this phosphorylation can occur at the locus of a hemostatic event. We also identify Ser362 as the major PKC phosphorylation site in vitronectin, and confirm this localization by means of synthetic peptides derived from the cluster of basic amino acids in vitronectin surrounding Ser362. We show that the PKC phosphorylation at Ser362 alters the functional properties of vitronectin, attenuating its cleavage by plasmin at Arg361-Ser362. This phosphorylation has the potential to regulate plasmin production from plasminogen by a feedback mechanism involving the above-mentioned plasmin cleavage, a loosening of the vitronectin grip on inhibitor 1 of plasminogen activators, and a subsequent latency of this regulatory inhibitor.

Genetic Analysis of the φX174 DNA Binding Protein

The φX174 J protein is 37 amino acids in length and contains 12 basic residues. There are no acidic amino acids in the protein. The basic residues are concentrated in two clusters in the N-terminus which are separated by a proline-rich region. To investigate the morphogenetic functions of the J protein and possible mechanisms by which it may bind DNA, a genetic analysis was conducted. Lysine → leucine and arginine → leucine substitutions were generated within the basic amino acid clusters. At least three substitutions were required to eliminate viabilityin vivo.Lethal mutants with three or four substitutions exhibit dominant lethal phenotypes, indicating that the mutant proteins retain enough function to interfere with productive assembly. In cells infected with a dominant lethal mutant, noninfectious packaged particles were produced. Infectivity can be restored by second-site suppressors in the viral coat protein which disrupt polar interactions atop the threefold axis of symmetry in the capsid. The viability of strains containing compensating frameshift mutations within the proline-rich region suggests that only the proline residues in this segment are critical for efficient function.

Distinct Troponin T Genes Are Expressed in Embryonic/Larval Tail Striated Muscle and Adult Body Wall Smooth Muscle of Ascidian

During development of the ascidian Halocynthia roretzi, the tadpole larva hatched from the tailbud embryo metamorphoses to the sessile adult with a body wall muscle. Although the adult body wall muscle is morphologically nonsarcomeric smooth muscle, it contains troponin complex consisting of three subunits (T, I, and C) as do vertebrate striated muscles. Different from vertebrate troponins, however, the smooth muscle troponin promotes actomyosin Mg2+-ATPase activity in the presence of high concentration of Ca2+, and this promoting property is attributable to troponin T. To address whether the embryonic/larval tail striated muscle and the adult smooth muscle utilize identical or different regulatory machinery, we cloned troponin T cDNAs from each cDNA library. The embryonic and the adult troponin Ts were encoded by distinct genes and shared only <60% identity with each other. Northern blotting and whole mount in situ hybridization revealed that these isoforms were specifically expressed in the embryonic/larval tail striated muscle and the adult smooth muscle, respectively. These results may imply that these isoforms regulate actin-myosin interaction in different manners. The adult troponin T under forced expression in mouse fibroblasts was unexpectedly located in the nuclei. However, a truncated protein with a deletion including a cluster of basic amino acids colocalized with tropomyosin on actin filaments. Thus, complex formation with troponin I and C immediately after the synthesis is likely to be essential for the protein to properly localize on the thin filaments.

Rin, a Neuron-Specific and Calmodulin-Binding Small G-Protein, and Rit Define a Novel Subfamily of Ras Proteins

cDNAs encoding two novel 25 kDa Ras-like proteins, Rit and Rin, were isolated from mouse retina using a degenerate PCR-based cloning strategy. Using the expressed sequence tag database, human orthologs were also obtained and sequenced. The protein sequences of Rit and Rin, which are 64% identical, are more similar to each other than to any known Ras protein. Their closest homologs in the databases are Mucor racemosus Ras2 and Ras3, to which they show approximately 48% identity. Rit and Rin both bind GTP in vitro. An unusual feature of their structure is that they lack a known recognition signal for C-terminal lipidation, a modification that is generally necessary for plasma membrane association among the Ras subfamily of proteins. Nonetheless, transiently expressed Rit and Rin are plasma membrane-localized. Both proteins contain a C-terminal cluster of basic amino acids, which could provide a mechanism for membrane association. Deletion analysis suggested that this region is important for Rit membrane binding but is not necessary for Rin. Rit, like most Ras-related proteins, is ubiquitously expressed. Rin, however, is unusual in that it is expressed only in neurons. In addition, Rin binds calmodulin through a C-terminal binding motif. These results suggest that Rit and Rin define a novel subfamily of Ras-related proteins, perhaps using a new mechanism of membrane association, and that Rin may be involved in calcium-mediated signaling within neurons.

Comparative mutagenesis of nuclear localization signals reveals the importance of neutral and acidic amino acids

Nuclear proteins contain information within their primary structures which causes them to accumulate selectively in the nucleus [1,2] by associating with the cytosolic receptor importin [3]. The α subunit of importin binds the nuclear localization signal (NLS), and the β subunit docks at the nuclear pore complex. The NLS of the simian virus 40 large T-antigen (SV40 T-ag) is a single cluster of basic amino acids (PKKKRKV132; single-letter code, the basic amino acids are shown in bold; [4,5]), whereas the NLS of nucleoplasmin is bipartite. The nucleoplasmin NLS requires two essential clusters of basic amino acids, separated by a mutation-tolerant spacer (KRPAATKKAGQAKKKK171; [6,7]). A SwissProt database search shows that more than 50% of nuclear proteins contain a match to this consensus, and many NLSs have since been found to conform to this type of motif in yeast, plants and animals [8–10]. A different NLS (PAAKRVKLD) has been reported in the oncoprotein c-Myc, but it has received little attention because, unlike other known NLSs, only three of nine residues are basic [11], and one residue is even acidic. Here, we report that constructs containing an inactive basic cluster downstream of the bipartite signal of nucleoplasmin can be directed to the nucleus by flanking them with specific neutral and acidic residues taken from the signal reported for c-Myc. Nuclear targeting by the single cluster KKKK is dependent on it being preceded by PAA and is stimulated if it is followed by the dipeptide LD. The relative positions of these elements are crucial to the function of these NLSs. All regions of the unconventional signal of c-Myc are functionally important. Contrary to conventional views, neutral and even acidic amino acids can play crucial roles in NLSs.

The sorting sequence of the peroxisomal integral membrane protein PMP47 is contained within a short hydrophilic loop.

No targeting sequence for peroxisomal integral membrane proteins has yet been identified. We have previously shown that a region of 67 amino acids is necessary to target Pmp47, a protein that spans the membrane six times, to peroxisomes. This region comprises two membrane spans and the intervening loop. We now demonstrate that the 20 amino acid loop, which is predicted to face the matrix, is both necessary and sufficient for peroxisomal targeting. Sufficiency was demonstrated with both chloramphenicol acetyltransferase and green fluorescent protein as carriers. There is a cluster of basic amino acids in the middle of the loop that we predict protrudes from the membrane surface into the matrix by a flanking stem structure. We show that the targeting signal is composed of this basic cluster and a block of amino acids immediately down-stream from it.

Specific Interaction of Topoisomerase IIβ and the CD3ε Chain of the T Cell Receptor Complex

T cell antigen receptor (TCR)-CD3 complex is composed of six different subunits: TCR alpha and TCR beta and CD3 gamma, CD3 delta, CD3 epsilon, and CD3 eta. Antigen recognition signals are transduced from TCR to the cytoplasm through the cytoplasmic domain of the CD3 chains. To understand the downstream signal transduction pathways, we cloned genes encoding proteins capable of binding to CD3 epsilon with a probe of glutathione S-transferase fused to the cytoplasmic region of CD3 epsilon. One of these clones was found to encode topoisomerase II beta (topoII beta). The binding region of CD3 epsilon is located within the N-terminal 12 amino acids containing the motif of a basic amino acid cluster. A similar motif was found in the gamma chain of Fc receptors (FcR gamma) but not in the CD33 eta chain, and indeed, FcR gamma but not CD3 eta bound to topoII beta. The binding region of topoII beta was determined to be the C terminus. Since this region appears to be the regulatory region of the enzymatic activity, the binding of CD3 epsilon might affect the function of topoII beta. Although topoII beta is localized mainly in the nucleus and CD3E is a membrane protein, we demonstrated the presence of CD3 epsilon in the nuclear fraction of thymocytes, which increased upon T cell activation. The specific interaction in cells was evidenced by co-immunoprecipitation of topoII beta and CD3E from the nuclear fraction of T cells. The possible function of this interaction is discussed.

Mutations of basic amino acids of NCp7 of human immunodeficiency virus type 1 affect RNA binding in vitro.

The nucleocapsid (NC) protein of human immunodeficiency virus type 1 is required for packaging of viral RNA and for virion assembly. It contains two clusters of basic amino acids, consisting of five and four amino acid residues, flanking the first of its two zinc fingers. These amino acid residues have been mutagenized to neutral ones individually, as well as in various combinations, by site-directed mutagenesis. Wild-type NCp7 and the mutant proteins were expressed as recombinant proteins in Escherichia coli, with six histidines as tags at their amino termini in order to allow efficient purification. The purified proteins were analyzed for RNA binding in vitro with human immunodeficiency virus type 1 5' leader RNA transcribed in vitro. Assays comprised Northwestern blots at various salt concentrations and filter binding tests which allowed determination of the dissociation constants of the various mutants. The results indicated that mutations of the amino acid R-7 and of R-32 and K-33 were more critical for RNA binding than other mutations. Mutation of the other amino acid residues reduced the binding affinity in proportion to the number of mutations. Mutation of seven of the nine basic amino acid residues reduced the binding of RNA by 50- to 90-fold.

COOH-terminal sequence motifs target the T cell protein tyrosine phosphatase to the ER and nucleus.

The noncatalytic domain of the human T cell protein tyrosine phosphatase (TCPTP) is alternatively spliced to generate a 45-kD form, p45TC, and a 48-kD form, p48TC (Champion-Arnaud et al., 1991; Mosinger et al., 1992). This manuscript concerns structural motifs in the noncatalytic segment of the enzyme responsible for targeting the two forms to different subcellular compartments. Endogenous and transiently expressed p48TC associates with the ER, as determined by sucrose gradient fractionation and indirect immunofluorescence, respectively. By contrast, p45TC localizes in the nucleus even though upon cell lysis it is not retained and fractionates with markers for soluble enzymes. Using fusion proteins consisting of beta-galactosidase and COOH-terminal fragments of p48TC, two motifs necessary for ER retention within a 70-residue targeting segment have been identified. These include the terminal 19 hydrophobic residues which comprise a potential membrane-spanning segment and residues 346-358 which encompass a cluster of basic amino acids that may represent another type of ER retention motif. The sequence RKRKR, which immediately precedes the splice junction, functions as a nuclear localization signal for p45TC.

The conserved, hydrophilic and arginine-rich N-terminal domain of cucumovirus coat proteins contributes to their anomalous electrophoretic mobilities in sodium dodecylsulfate-polyacrylamide gels

Although the Mr values of the coat proteins (CPs) of several cucumoviruses have been calculated from their deduced amino acid sequences to be approximately 24,000, the experimentally determined M(r) values using the Laemmli SDS-PAGE system were 30,000-31,000. Examination of the amino acid composition revealed that these CPs are neither highly acidic nor highly basic. Post-translational glycosylation or phosphorylation were also ruled out as contributing factors to the observed anomalous electrophoretic mobility because the products of in vitro translation of cucumovirus RNA 4 and in vivo bacterial expression of the cloned CP gene co-migrated with authentic cucumovirus CPs. Comparison of the hydropathy profiles of the CPs revealed the presence in each of a strikingly similar, highly hydrophilic N-terminal domain of 30-32 amino acid residues that contains a cluster of basic amino acids, mainly arginine. Selective chemical cleavage at tryptophan residues in the CPs of cucumoviruses, known to contain single tryptophan residues, yielded two peptides; an N-terminal peptide that contained the conserved hydrophilic domain and a C-terminal peptide. SDS-PAGE analysis showed that the N-terminal, but not the C-terminal, peptide exhibited the anomalous electrophoretic mobility.

Neuroglycan C, a Novel Membrane-spanning Chondroitin Sulfate Proteoglycan That Is Restricted to the Brain

Monoclonal antibodies were raised to membrane-bound proteoglycans derived from rat brain, and four monoclonal antibodies that recognized a 150-kDa chondroitin sulfate proteoglycan with a core glycoprotein of 120 kDa were obtained. Immunohistological study revealed that the proteoglycan was associated with developing neurons. We screened rat brain cDNA libraries using the four monoclonal antibodies and isolated overlapping cDNA clones that encoded the entire core protein of 514 amino acids plus a 30-residue signal peptide. The deduced amino acid sequence suggested an integral membrane protein divided into five structurally different domains: an N-terminal domain to which chondroitin sulfate chains might be attached, a basic amino acid cluster consisting of seven arginine and two lysine residues, a cysteine-containing domain, a membrane-spanning segment, and a C-terminal cytoplasmic domain of 95 amino acids. On Northern blots, the cDNA hybridized with a single mRNA of 3.1 kilobases that was detectable in brains of neonatal and adult rats but not in kidney, liver, lung, and muscle of either. The sequence of the proteoglycan did not exhibit significant homology to any other known protein, indicating that the proteoglycan, designated neuroglycan C, is a novel integral membrane proteoglycan.

Analysis of Putative Heparin-binding Domains of Fibroblast Growth Factor-1: USING SITE-DIRECTED MUTAGENESIS AND PEPTIDE ANALOGUES

The contribution of individual basic amino acids within three putative "consensus sequences" for heparin binding of fibroblast growth factor-1 have been examined by site-directed mutagenesis. The results indicate that a significant reduction in the apparent affinity of fibroblast growth factor-1 for heparin is only observed when basic residues in one of the three regions are mutated. Mutation in the other regions are without affect on heparin binding. The heparin binding properties of synthetic peptides based on the three "consensus sequences" paralleled the mutagenesis results. That is, synthetic peptides corresponding to regions of the protein that were affected by mutagenesis with respect to heparin binding exhibited a relatively high affinity for immobilized heparin, whereas those corresponding to regions of similar charge density that were unaffected by mutagenesis did not. In addition, amino acid substitution of a nonbasic residue in the heparin-binding peptide could abolish its heparin binding capacity. The heparin-binding peptide could antagonize the mitogenic activity of FGF-1, probably because of the heparin dependence of this activity. Together these data demonstrate that the heparin binding properties of fibroblast growth factor-1 are dictated by structural features more complex than clusters of basic amino acids. The results of these and other studies indicate that consensus motifs for heparin-binding require further definition. More importantly, the results provide a basis for the design of peptide-based inhibitors of FGF-1.

Interferon Gamma Binds to Extracellular Matrix Chondroitin-Sulfate Proteoglycans, Thus Enhancing Its Cellular Response

The amino acid sequence of interferon gamma (IFN-gamma) has basic amino acid clusters similar to the heparin-binding consensus sequences found in other proteins that bind to proteoglycans (PGs). We investigated whether recombinant human IFN-gamma could bind to extracellular matrix (ECM) PGs secreted by human arterial smooth muscle cells (HASMCs) in vitro and whether the interaction affected the cellular response to IFN-gamma. As an in vitro model of ECM we used the basement membrane from HASMCs in culture. The binding of 125I-IFN-gamma to ECM was reduced significantly by pretreatment of ECM with chondroitinase ABC, an enzyme that degrades chondroitin-sulfate glycosaminoglycans. IFN-gamma binding to ECM was reduced by increasing concentrations of chondroitin-6-sulfate. 125I-IFN-gamma (0.05 to 2 ng/mL) binding data indicated an apparent Kd of 2 x 10(-11) mol/L and a maximum binding of 1.6 x 10(6) IFN-gamma molecules bound per square millimeter of ECM. Experiments with synthetic peptides suggested that residues 127 through 135 (AKTGKRKRS) are involved in the binding. The binding to chondroitin-sulfate PGs was confirmed by affinity chromatography of isolated [35S]chondroitin-sulfate PGs from ECM and cell-culture medium on immobilized IFN-gamma. The binding was abolished by treatment with chondroitinase ABC. ECM-bound IFN-gamma was more effective in inducing the expression of class II major histocompatibility antigens such as HLA-DR in HASMCs and human arterial endothelial cells than soluble IFN-gamma. These results suggest a role for chondroitin-sulfate PGs in immobilizing IFN-gamma in the ECM compartment and enhancing the cellular response to IFN-gamma.

Expression cloning of a human polysialyltransferase that forms the polysialylated neural cell adhesion molecule present in embryonic brain.

Polysialic acid is a developmentally regulated posttranslational modification of the neural cell adhesion molecule (N-CAM). It has been suggested that this large anionic carbohydrate modulates the adhesive property of N-CAM, but the precise function of polysialic acid is not known. Here we describe the isolation and functional expression of a cDNA encoding a human polysialyltransferase. For this expression cloning, COS-1 cells were cotransfected with a human fetal brain cDNA library and a cDNA encoding human N-CAM. Transfected COS-1 cells were stained with a monoclonal antibody specific for polysialic acid and enriched by fluorescence-activated cell sorting. Sibling selection of recovered plasmids resulted in a cDNA clone that directs the expression of polysialic acid on the cell surface. The deduced amino acid sequence indicates that the polysialyltransferase shares a common sequence motif with other sialyltransferases cloned so far. The polysialyltransferase is, however, distinct by having two clusters of basic amino acids. The amount of the polysialyltransferase transcripts correlates well with the formation of polysialic acid in various human tissues, and is abundant in the fetal brain but not in the adult brain. Moreover, HeLa cells stably expressing polysialic acid and N-CAM promoted neurite outgrowth and sprouting. These results indicate that the cloned polysialyltransferase forms polysialylated, embryonic N-CAM, which is critical for plasticity of neural cells.

Tissue factor pathway inhibitor

After intravenous and subcutaneous injection, heparins and low-molecular-weight heparins release tissue factor pathway inhibitor (TFPI) into the blood stream. Protamine immediately reverses the plasma levels of TFPI to pretreatment values. A low-molecular-weight dermatan sulphate releases only very small amounts of TFPI after intravenous injection without a clear dose-dependent effect. Together with other data from literature, it seems likely that TFPI is rather specifically released by acidic glycosaminoglycans. In the present paper a binding region for heparins is reported to be located in the C-terminal end. A basic amino acid cluster occurs between 256-lysine and 261-lysine. This proposed heparin recognition region in TFPI is similar to the recognition region in antithrombin III and other proteins. The binding between the basic region at the C-terminal end of TFPI and the negatively charged sulphate or carboxyl-groups of glycosaminoclycans may occur in a linear manner. A comparison of a helical wheel diagram of antithrombin III and tissue factor pathway inhibitor support also the proposal of this form of a heparin recognition region in TFPI. Further studies are now required to analyse these interactions.

Identification and Characterization of a Novel Cytokine-inducible Nuclear Protein from Human Endothelial Cells

Vascular endothelial cells undergo profound changes upon cellular activation including expression of a spectrum of cell activation-associated genes. These changes play important roles in many physiological and pathological events. By differential screening of a cDNA library prepared from interleukin-1 alpha and tumor necrosis factor-alpha-stimulated human dermal microvascular endothelial cells, we have identified a novel cytokine-inducible gene, designated as C-193. The compiled cDNA sequence of C-193 is 1901 base pairs long and shows no significant homology with any known gene sequence. Genomic DNA analysis revealed that C-193 is encoded by a single gene, which is conserved in different mammalian species. The C-193 gene was localized to human chromosome 10 by Southern blot analysis of somatic cell hybrids. Multiple AT-rich mRNA decay elements were identified in the 3'-untranslated region. C-193 mRNA expression was rapidly and transiently induced by treatment with interleukin-1 alpha or tumor necrosis factor-alpha, reached a peak of expression about 16 h post tumor necrosis factor-alpha stimulation, and the induction of C-193 was protein synthesis independent. Lipopolysaccharide and cycloheximide were also potent inducers of C-193 mRNA. Therefore, C-193 represents a new addition to the primary response gene family. In vitro translation of C-193 yielded a 36-kDa protein product, consistent with the predicted open reading frame of 318 amino acids and a calculated molecular mass of 36 kDa for C-193 protein. The predicted protein sequence contains a basic amino acid cluster similar to a nuclear localization signal, four tandem repeats of ankyrin-like sequence, and multiple consensus protein phosphorylation sites. C-193 was engineered with a FLAG tag at its carboxyl terminus and transiently expressed in COS cells. Consistent with the presence of a putative nuclear localization signal, the C-193-FLAG protein was localized to the nucleus of transfected COS cells by indirect immunofluorescence microscopy. C-193-FLAG prepared in vitro was capable of binding DNA cellulose. These results indicate that C-193 protein may play an important role in endothelial cell activation.

Amino Acid Sequence and Disulphide-bridge Pattern of three gamma-Thionins from Sorghum bicolor

The complete primary structure of a new alpha-amylase inhibitor from Sorghum bicolor belonging to the gamma-thionin family has been determined and the amino acid sequences of two components of the family already elucidated have been corrected by combining the classical Edman degradation with advanced mass spectrometric procedures. The same integrated approach allowed us to define the pattern of the disulphide bridges in the three isoinhibitors. The arrangement of the cysteine pairing was determined as Cys3-Cys47, Cys14-Cys34, Cys20-Cys41 and Cys24-Cys43. The amino acid sequences of the alpha-amylase inhibitors share a high degree of similarity with the related plant gamma-thionins. All these proteins consist of 47 residues, contain eight cysteine residues forming four disulphide bridges, and show the presence of two clusters of basic amino acids located at both ends of the polypeptide chain. The pattern of S-S bridges determined for the isoinhibitors is identical to that inferred by NMR analysis in two related gamma-thionins, thus suggesting a highly conserved organization of the disulphide pairing. These results indicate that the structural similarities among the different gamma-thionins extend far beyond the primary structure and possibly concern the secondary structure and the general folding of the entire gamma-thionin family.

The primary structure of D-amino acid oxidase from Rhodotorula gracilis

The amino acid sequence of D-amino acid oxidase from Rhodotorula gracilis was determined by automated Edman degradation of peptides generated by enzymatic and chemical cleavage. The enzyme monomer contains 368 amino acid residues and its sequence is homologous to that of other known D-amino acid oxidases. Six highly conserved regions appear to have a specific role in binding of coenzyme FAD, in active site topology and in peroxisomal targeting. Moreover, Rhodotorula gracilis D-amino acid oxidase contains a region with a cluster of basic amino acids, probably exposed to solvent, which is absent in other D-amino acid oxidases.

Nuclear targeting of the tegument protein pp65 (UL83) of human cytomegalovirus: an unusual bipartite nuclear localization signal functions with other portions of the protein to mediate its efficient nuclear transport

Large amounts of pp65 (UL83) of human cytomegalovirus are translocated to the cell nucleus during the first minutes after uptake of the tegument protein from infecting viral particles. Two stretches of basic amino acids which resembled nuclear localization signals (NLS) of both the simian virus 40 type and the bipartite type were found in the primary structure of pp65. Deletion of these sequences significantly impaired nuclear localization of the truncated proteins after transient expression. The results indicated that both elements contributed to the nuclear localization of the protein. When fused to the bacterial beta-galactosidase, only one of the two basic elements was sufficient to mediate nuclear translocation. This element consisted of two clusters of basic amino acids (boxes C and D), which were separated by a short spacer sequence. In contrast to other bipartite NLS of animal cells, both basic boxes C and D functioned independently in nuclear transport, thus resembling simian virus 40-type NLS. Yet, complete translocation of beta-galactosidase was only found in the bipartite configuration. When both boxes C and D were fused, thereby deleting the intervening sequences, the nuclear transport of beta-galactosidase was reduced to levels seen with constructs in which only one of the boxes was present. Appropriate spacing, therefore, was important but not absolutely required. This was in contrast with results for other bipartite NLS, in which spacer deletions led to complete cytoplasmic retention. The presented results demonstrate that efficient nuclear transport of pp65 is mediated by one dominant NLS and additional targeting sequences. The major NLS of pp65 is an unusual signal sequence composed of two weak NLS which function together as one strong bipartite nuclear targeting signal.

A cluster of basic amino acids within an alpha-helix is essential for alpha-subunit recognition by the glycoprotein hormone N-acetylgalactosaminyltransferase.

The glycoprotein hormone N-acetylgalactosaminyltransferase is responsible for synthesis of Asn-linked oligosaccharides terminating with GalNAc-4-SO4 on lutropin, thyrotropin, and the uncombined glycoprotein hormone alpha subunit. We previously established that a recognition determinant for the N-acetylgalactosaminyltransferase is contained within a 22-amino acid glycopeptide fragment of the alpha subunit. We proposed that the tripeptide Pro-Leu-Arg is an essential element of the recognition determinant. Using site-directed mutagenesis we have examined the role of individual amino acids in recognition by the glycoprotein hormone N-acetylgalactosaminyltransferase. Within the sequence Pro40-Leu41-Arg42-Ser43-Lys44-Lys45, Lys44, and Lys45, as well as Arg42 of the tripeptide, are essential for recognition. Substitution of the Leu41 with other amino acids can either increase or decrease the rate of GalNAc transfer over an 8-fold range, suggesting that the middle amino acid of the tripeptide plays a modulatory role in recognition. The critical Leu41-Arg42 and Lys44-Lys45 residues are present on the same surface of an alpha-helix, which projects from the surface of the alpha subunit. Our results indicate that an essential element of the recognition determinant consists of a cluster of basic residues and that neutral but not negatively charged residues are tolerated within this cluster.