Additional Protein Domains Research Articles

The Essential Phosphoinositide Kinase MSS-4 Is Required for Polar Hyphal Morphogenesis, Localizing to Sites of Growth and Cell Fusion in Neurospora crassa

Fungal hyphae and plant pollen tubes are among the most highly polarized cells known and pose extraordinary requirements on their cell polarity machinery. Cellular morphogenesis is driven through the phospholipid-dependent organization at the apical plasma membrane. We characterized the contribution of phosphoinositides (PIs) in hyphal growth of the filamentous ascomycete Neurospora crassa. MSS-4 is an essential gene and its deletion resulted in spherically growing cells that ultimately lyse. Two conditional mss-4-mutants exhibited altered hyphal morphology and aberrant branching at restrictive conditions that were complemented by expression of wild type MSS-4. Recombinant MSS-4 was characterized as a phosphatidylinositolmonophosphate-kinase phosphorylating phosphatidylinositol 4-phosphate (PtdIns4P) to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). PtdIns3P was also used as a substrate. Sequencing of two conditional mss-4 alleles identified a single substitution of a highly conserved Y750 to N. The biochemical characterization of recombinant protein variants revealed Y750 as critical for PI4P 5-kinase activity of MSS-4 and of plant PI4P 5-kinases. The conditional growth defects of mss-4 mutants were caused by severely reduced activity of MSS-4(Y750N), enabling the formation of only trace amounts of PtdIns(4,5)P2. In N. crassa hyphae, PtdIns(4,5)P2 localized predominantly in the plasma membrane of hyphae and along septa. Fluorescence-tagged MSS-4 formed a subapical collar at hyphal tips, localized to constricting septa and accumulated at contact points of fusing N. crassa germlings, indicating MSS-4 is responsible for the formation of relevant pools of PtdIns(4,5)P2 that control polar and directional growth and septation. N. crassa MSS-4 differs from yeast, plant and mammalian PI4P 5-kinases by containing additional protein domains. The N-terminal domain of N. crassa MSS-4 was required for correct membrane association. The data presented for N. crassa MSS-4 and its roles in hyphal growth are discussed with a comparative perspective on PI-control of polar tip growth in different organismic kingdoms.

Origin and Diversification of TRIM Ubiquitin Ligases

Most proteins of the TRIM family (also known as RBCC family) are ubiquitin ligases that share a peculiar protein structure, characterized by including an N-terminal RING finger domain closely followed by one or two B-boxes. Additional protein domains found at their C termini have been used to classify TRIM proteins into classes. TRIMs are involved in multiple cellular processes and many of them are essential components of the innate immunity system of animal species. In humans, it has been shown that mutations in several TRIM-encoding genes lead to diverse genetic diseases and contribute to several types of cancer. They had been hitherto detected only in animals. In this work, by comprehensively analyzing the available diversity of TRIM and TRIM-like protein sequences and evaluating their evolutionary patterns, an improved classification of the TRIM family is obtained. Members of one of the TRIM subfamilies defined, called Subfamily A, turn to be present not only in animals, but also in many other eukaryotes, such as fungi, apusozoans, alveolates, excavates and plants. The rest of subfamilies are animal-specific and several of them originated only recently. Subfamily A proteins are characterized by containing a MATH domain, suggesting a potential evolutionary connection between TRIM proteins and a different type of ubiquitin ligases, known as TRAFs, which contain quite similar MATH domains. These results indicate that the TRIM family emerged much earlier than so far thought and contribute to our understanding of its origin and diversification. The structural and evolutionary links with the TRAF family of ubiquitin ligases can be experimentally explored to determine whether functional connections also exist.

PMO-132 Histone variant macroh2a1 knock-out worsens high fat diet-induced systemic insulin resistance, glucose intolerance and hepatic steatosis in mice

IntroductionHepatic steatosis is a major risk factor for the development of severe liver damage, including fibrosis, cirrhosis and hepatocellular carcinoma. It often exists as a co-morbidity factor with diabetes type...

Iron induced eukaryotic initiation factor/ mRNA binding affinity change

Cellular iron metabolism is regulated by expression of iron responsive element (IRE) containing‐mRNA to control iron related protein levels (e.g. ferritin). The cytoplasmic iron regulatory proteins, IRPs, bind to iron response element (IRE) mRNAs, which represses IRE‐mRNA translation. IRE‐RNA binds to Fe2+ (in the absence of oxygen to prevent insoluble oxides) and the iron metabolite change the IRE‐RNA conformation resulting in decreased IRP (repressor) binding and increased eIF4F (enhancer) binding. eIF4F binds IRE‐RNA with high affinity (Kd=39.8±5.6nM) and further eIF4F: IRP competitively binds to IRE‐mRNA. eIF4G, a component of eIF4F, is also found to bind to the IRE‐RNA. The eIF4G middle domain binding affinity to the ferrtin IRE is lower than eIF4F and IRE binding doses not respond to metal concentration changes. While the eIF4G middle domain contains the RNA binding domain, additional protein domains and/or initiation factors (e.g. eIF4A, eIF4E) are necessary for functioning of the iron metabolite IRE‐ribo regulator.This work is supported in whole or in part, by National Institutes of Health Grants DK20251 (D.J.G. and E.C.T.) and National Science Foundation Grant MCB 0814051 (D.J.G.)

Functional characterization of the nuclear localization signal of OREBP/TonEBP

The Osmotic Response Element-Binding Protein (OREBP), also known as TonEBP or NFAT5, is a transcription factor that mediates cellular adaptations to extracellular hypertonic stress. OREBP is regulated at multiple levels including nuclear import. Previous studies suggested that the consensus nuclear localization signal (NLS, amino acid residues 199–216) was crucial for the nuclear import of OREBP. To better delineate the role of NLS in OREBP nuclear import, we generated and studied the sub-cellular localization of different PEPCK fusion proteins that contains the minimal consensus NLS alone, or different fragments that cover NLS and its flanking sequences. Minimal NLS alone failed to direct PEPCK to the nucleus. Instead, the nuclear import function of NLS is reconstituted by including two flanking domains that were located in the N- and C-terminal to the NLS. Alanine-scanning mutagenesis of the corresponding region using FLAG-tagged OREBP further revealed two clusters of amino acids that are responsible for its nuclear translocation. Thus, although a general feature of monopartite and bipartite NLS is the present of multiple basic residues, and is thought to be sufficient for directing nuclear import of transcription factors, our data suggested that the NLS of OREBP is by itself essential but insufficient to direct nuclear import, and additional protein domains are required for its function.

In Silico Identification of Carboxylate Clamp Type Tetratricopeptide Repeat Proteins in Arabidopsis and Rice As Putative Co-Chaperones of Hsp90/Hsp70

The essential eukaryotic molecular chaperone Hsp90 operates with the help of different co-chaperones, which regulate its ATPase activity and serve as adaptors to recruit client proteins and other molecular chaperones, such as Hsp70, to the Hsp90 complex. Several Hsp90 and Hsp70 co-chaperones contain the tetratricopeptide repeat (TPR) domain, which interacts with the highly conserved EEVD motif at the C-terminal ends of Hsp90 and Hsp70. The acidic side chains in EEVD interact with a subset of basic residues in the TPR binding pocket called a ‘carboxylate clamp’. Since the carboxylate clamp residues are conserved in the TPR domains of known Hsp90/Hsp70 co-chaperones, we carried out an in silico search for TPR proteins in Arabidopsis and rice comprising of at least one three-motif TPR domain with conserved amino acid residues required for Hsp90/Hsp70 binding. This approach identified in Arabidopsis a total of 36 carboxylate clamp (CC)-TPR proteins, including 24 novel proteins, with potential to interact with Hsp90/Hsp70. The newly identified CC-TPR proteins in Arabidopsis and rice contain additional protein domains such as ankyrin, SET, octicosapeptide/Phox/Bem1p (Phox/PB1), DnaJ-like, thioredoxin, FBD and F-box, and protein kinase and U-box, indicating varied functions for these proteins. To provide proof-of-concept of the newly identified CC-TPR proteins for interaction with Hsp90, we demonstrated interaction of AtTPR1 and AtTPR2 with AtHsp90 in yeast two-hybrid and in vitro pull down assays. These findings indicate that the in silico approach used here successfully identified in a genome-wide context CC-TPR proteins with potential to interact with Hsp90/Hsp70, and further suggest that the Hsp90/Hsp70 system relies on TPR co-chaperones more than it was previously realized.

Combinatorial Enzyme Design Probes Allostery and Cooperativity in the Trypsin Fold

Converting one enzyme into another is challenging due to the uneven distribution of important amino acids for function in both protein sequence and structure. We report a strategy for protein engineering allowing an organized mixing and matching of genetic material that leverages lower throughput with increased quality of screens. Our approach successfully tested the contribution of each surface-exposed loop in the trypsin fold alone and the cooperativity of their combinations towards building the substrate selectivity and Na+-dependent allosteric activation of the protease domain of human coagulation factor Xa into a bacterial trypsin. As the created proteases lack additional protein domains and protein co-factor activation mechanism requisite for the complexity of blood coagulation, they are stepping-stones towards further understanding and engineering of artificial clotting factors.

Knotted Fusion Proteins Reveal Unexpected Possibilities in Protein Folding

Proteins that contain a distinct knot in their native structure are impressive examples of biological self-organization. Although this topological complexity does not appear to cause a folding problem, the mechanisms by which such knotted proteins form are unknown. We found that the fusion of an additional protein domain to either the amino terminus, the carboxy terminus, or to both termini of two small knotted proteins did not affect their ability to knot. The multidomain constructs remained able to fold to structures previously thought unfeasible, some representing the deepest protein knots known. By examining the folding kinetics of these fusion proteins, we found evidence to suggest that knotting is not rate limiting during folding, but instead occurs in a denatured-like state. These studies offer experimental insights into when knot formation occurs in natural proteins and demonstrate that early folding events can lead to diverse and sometimes unexpected protein topologies.

Evolutionary Analysis of the TPP-Dependent Enzyme Family

The evolutionary relationships of the thiamine pyrophosphate (TPP)-dependent family of enzymes was investigated by generation of a neighbor joining phylogenetic tree using sequences from the conserved pyrophosphate (PP) and pyrimidine (Pyr) binding domains of 17 TPP-dependent enzymes. This represents the most comprehensive analysis of TPP-dependent enzyme evolution to date. The phylogeny was shown to be robust by comparison with maximum likelihood trees generated for each individual enzyme and also broadly confirms the evolutionary history proposed recently from structural comparisons alone (Duggleby 2006). The phylogeny is most parsimonious with the TPP enzymes having arisen from a homotetramer which subsequently diverged into an alpha(2)beta(2) heterotetramer. The relationship between the PP- and Pyr-domains and the recruitment of additional protein domains was examined using the transketolase C-terminal (TKC)-domain as an example. This domain has been recruited by several members of the family and yet forms no part of the active site and has unknown function. Removal of the TKC-domain was found to increase activity toward beta-hydroxypyruvate and glycolaldehyde. Further truncations of the Pyr-domain yielded several variants with retained activity. This suggests that the influence of TKC-domain recruitment on the evolution of the mechanism and specificity of transketolase (TK) has been minor, and that the smallest functioning unit of TK comprises the PP- and Pyr-domains, whose evolutionary histories extend to all TPP-dependent enzymes.

DMi-2 Chromatin Binding and Remodeling Activities Are Regulated by dCK2 Phosphorylation

A plethora of ATP-dependent chromatin-remodeling enzymes have been identified during the last decade. Many have been shown to play pivotal roles in the organization and expression of eukaryotic genomes. It is clear that their activities need to be tightly regulated to ensure their coordinated action. However, little is known about how ATP-dependent remodelers are regulated at the molecular level. Here, we have investigated the ATP-dependent chromatin remodeling enzyme Mi-2 of Drosophila melanogaster. Radioactive labeling of S2 cells reveals that dMi-2 is a phosphoprotein in vivo. dMi-2 phosphorylation is constitutive, and we identify dCK2 as a major dMi-2 kinase in cell extracts. dCK2 binds to and phosphorylates a dMi-2 N-terminal region. Dephosphorylation of recombinant dMi-2 increases its affinity for the nucleosome substrate, nucleosome-stimulated ATPase, and ATP-dependent nucleosome mobilization activities. Our results reveal a potential mechanism for regulation of the dMi-2 enzyme and point toward CK2 phosphorylation as a common feature of CHD family ATPases.

The Phosphoinositide-3-phosphatase MTMR2 Associates with MTMR13, a Membrane-associated Pseudophosphatase Also Mutated in Type 4B Charcot-Marie-Tooth Disease

Charcot-Marie-Tooth disease type 4B (CMT4B) is a severe, demyelinating peripheral neuropathy characterized by distinctive, focally folded myelin sheaths. CMT4B is caused by recessively inherited mutations in either myotubularin-related 2 (MTMR2) or MTMR13 (also called SET-binding factor 2). MTMR2 encodes a member of the myotubularin family of phosphoinositide-3-phosphatases, which dephosphorylate phosphatidylinositol 3-phosphate (PI(3)P) and bisphosphate PI(3,5)P2. MTMR13 encodes a large, uncharacterized member of the myotubularin family. The MTMR13 phosphatase domain is catalytically inactive because the essential Cys and Arg residues are absent. Given the genetic association of both MTMR2 and MTMR13 with CMT4B, we investigated the biochemical relationship between these two proteins. We found that the endogenous MTMR2 and MTMR13 proteins are associated in human embryonic kidney 293 cells. MTMR2-MTMR13 association is mediated by coiled-coil sequences present in each protein. We also examined the cellular localization of MTMR2 and MTMR13 using fluorescence microscopy and subcellular fractionation. We found that (i) MTMR13 is a predominantly membrane-associated protein; (ii) MTMR2 and MTMR13 cofractionate in both a light membrane fraction and a cytosolic fraction; and (iii) MTMR13 membrane association is mediated by the segment of the protein which contains the pseudophosphatase domain. This work, which describes the first cellular or biochemical investigation of the MTMR13 pseudophosphatase protein, suggests that MTMR13 functions in association with MTMR2. Loss of MTMR13 function in CMT4B2 patients may lead to alterations in MTMR2 function and subsequent alterations in 3-phosphoinositide signaling. Such a mechanism would explain the strikingly similar phenotypes of patients with recessive mutations in either MTMR2 or MTMR13.

Sorting nexin 17 facilitates LRP recycling in the early endosome

The low-density lipoprotein (LDL) receptor-related protein (LRP) is a multiligand endocytic receptor and a member of the LDL receptor family. Here we show that sorting nexin 17 (Snx 17) is part of the cellular sorting machinery that regulates cell surface levels of LRP by promoting its recycling. While the phox (PX) domain of Snx 17 interacts with phosphatidylinositol-3-phosphate for membrane association, the FERM domain and the carboxyl-terminal region participate in LRP binding. Immunoelectron microscopy shows that the membrane-bound fraction of Snx 17 is localized to the limiting membrane and recycling tubules of early endosomes. The NPxY motif, proximal to the plasma membrane in the LRP cytoplasmic tail, is identified as the Snx 17-binding motif. Functional mutation of this motif did not interfere with LRP endocytosis, but decreased LRP recycling from endosomes, resulting in increased lysosomal degradation. Similar effects are found after knockdown of endogenous Snx 17 expression by short interfering RNA. We conclude that Snx 17 binds to a motif in the LRP tail distinct from the endocytosis signals and promotes LRP sorting to the recycling pathway in the early endosomes.

The translational apparatus of Chlamydomonas reinhardtii chloroplast

Genetic and biochemical studies have revealed that chloroplast gene expression in Chlamydomonas is controlled primarily post-transcriptionally, including events that effect mRNA processing and stability, and during the translation of plastid mRNAs into proteins. Many of the proteins required for chloroplast gene expression are encoded in the nuclear genome, and most of these proteins have yet to be identified biochemically. Emergence of the draft sequence of the Chlamydomonas nuclear genome has enabled us to carry out a prediction and comparative analysis of the proteins required for chloroplast mRNA translation. Putative translation factor genes have been identified by homology search, and functional chloroplast ribosomal protein genes have been compiled based on our recent proteomic studies. This bioinformatic and proteomic analysis shows that the translational apparatus of Chlamydomonas is related to that of bacteria, but is more complex. Chlamydomonas chloroplasts contain all of the general translation factors found in bacteria, and a majority of the ribosomal proteins are conserved between plastids and bacteria. However, Chlamydomonas contains a number of additional proteins and protein domains associated with the plastid ribosome, while some ribosomal proteins are either quite divergent or lacking. In addition, Chlamydomonas chloroplasts contain a number of mRNA specific translation factors that are not found in bacteria.

Frequent alternative splicing of human genes.

Alternative splicing can produce variant proteins and expression patterns as different as the products of different genes, yet the prevalence of alternative splicing has not been quantified. Here the spliced alignment algorithm was used to make a first inventory of exon-intron structures of known human genes using EST contigs from the TIGR Human Gene Index. The results on any one gene may be incomplete and will require verification, yet the overall trends are significant. Evidence of alternative splicing was shown in 35% of genes and the majority of splicing events occurred in 5' untranslated regions, suggesting wide occurrence of alternative regulation. Most of the alternative splices of coding regions generated additional protein domains rather than alternating domains.

Type IIA procollagen containing the cysteine-rich amino propeptide is deposited in the extracellular matrix of prechondrogenic tissue and binds to TGF-beta1 and BMP-2.

Type II procollagen is expressed as two splice forms. One form, type IIB, is synthesized by chondrocytes and is the major extracellular matrix component of cartilage. The other form, type IIA, contains an additional 69 amino acid cysteine-rich domain in the NH2-propeptide and is synthesized by chondrogenic mesenchyme and perichondrium. We have hypothesized that the additional protein domain of type IIA procollagen plays a role in chondrogenesis. The present study was designed to determine the localization of the type IIA NH2-propeptide and its function during chondrogenesis. Immunofluorescence histochemistry using antibodies to three domains of the type IIA procollagen molecule was used to localize the NH2-propeptide, fibrillar domain, and COOH-propeptides of the type IIA procollagen molecule during chondrogenesis in a developing human long bone (stage XXI). Before chondrogenesis, type IIA procollagen was synthesized by chondroprogenitor cells and deposited in the extracellular matrix. Immunoelectron microscopy revealed type IIA procollagen fibrils labeled with antibodies to NH2-propeptide at approximately 70 nm interval suggesting that the NH2-propeptide remains attached to the collagen molecule in the extracellular matrix. As differentiation proceeds, the cells switch synthesis from type IIA to IIB procollagen, and the newly synthesized type IIB collagen displaces the type IIA procollagen into the interterritorial matrix. To initiate studies on the function of type IIA procollagen, binding was tested between recombinant NH2-propeptide and various growth factors known to be involved in chondrogenesis. A solid phase binding assay showed no reaction with bFGF or IGF-1, however, binding was observed with TGF-beta1 and BMP-2, both known to induce endochondral bone formation. BMP-2, but not IGF-1, coimmunoprecipitated with type IIA NH2-propeptide. Recombinant type IIA NH2-propeptide and type IIA procollagen from media coimmunoprecipitated with BMP-2 while recombinant type IIB NH2-propeptide and all other forms of type II procollagens and mature collagen did not react with BMP-2. Taken together, these results suggest that the NH2-propeptide of type IIA procollagen could function in the extracellular matrix distribution of bone morphogenetic proteins in chondrogenic tissue.

Different CD44 Splicing Patterns Define Prognostic Subgroups in Multiple Myeloma

CD44 variant isoforms (CD44v) are generated by alternative splicing of the nuclear RNA resulting in the expression of additional protein domains in the extracellular region of the CD44 standard molecule (CD44s). In multiple myeloma (MM), CD44 mediates binding of tumor cells to stroma and regulates interleukin-6 production. To evaluate the role of CD44v isoforms in MM, CD44v expression was analyzed by immunohistochemical staining of 64 bone marrow biopsies from 38 MM patients. Expression of variant isoforms containing the 9v domain was observed in 36% of cases and was associated with an advanced stage (P < .02; n = 61), a progressive disease (P < .001; n = 61), and a shorter overall survival (P < .02; n = 36). In contrast, 3v, 4v, 6v, or 10v isoforms were detected only in a small percentage of the patients. To analyze the exon composition in RNA-transcripts, reverse transcriptase polymerase chain reaction analyses followed by Southern hybridization with exon-specific probes were performed in fluorescence-activated cell sorted myeloma plasma cells. Tumors expressing the 9v domain showed complex, 9v-containing transcripts in combinations with the 3v, 7v, 8v, and 10v exons. Identical transcripts were detected in several myeloma cell lines and in a Ki-1 B-immunoblastic lymphoma. Similar to high-grade non-Hodgkin's lymphoma and gastric and renal cell carcinoma, overexpression of 9v-containing isoforms in MM is related to an unfavorable clinical presentation and represents a new prognostic parameter.

Elements of Neural Adhesion Molecules and a Yeast Vacuolar Protein Sorting Receptor Are Present in a Novel Mammalian Low Density Lipoprotein Receptor Family Member

Normal cell development depends to a large part on multifunctional proteins that have evolved by recombination of proven modular elements. We now have discovered and characterized in rabbit such a multi-domain protein, and classify it as novel member of the low density lipoprotein (LDL) receptor gene family. The extracellular portion of the approximately 250-kDa membrane protein, termed LR11, contains a cluster of 11 LDL receptor ligand binding repeats, a group of 5 LDL receptor "YWTD" repeats, a large hexarepeat domain of structural elements found in neural cell adhesion molecules, and a domain with similarity to a yeast receptor for vacuolar protein sorting, VPS10. The cytoplasmic domain exhibits features typical of endocytosis-competent coated-pit receptors. The mosaic, and presumably multifunctional, receptor is expressed abundantly in brain, in particular the hippocampus, dentate gyrus, and cerebral cortex, and is present at significant levels in liver, adrenal glands, and testis. Western blotting of tissues and ligand blotting of LR11-transfected cells demonstrated that the novel protein binds apolipoprotein E-containing lipoproteins. In contrast to the LDL receptor, hepatic expression of LR11 is unaffected by hyperlipidemia. The identification of this highly conserved and superbly complex protein offers the opportunity to gain new insights into the emergence of multifunctional mosaic proteins akin to the ever expanding LDL receptor gene family.

A molecular model for the redox potential difference between thioredoxin and DsbA, based on electrostatics calculations

The disulphide active sites of thioredoxin and DsbA are known to possess a high degree of structural homology. However, DsbA is a much stronger oxidant than thioredoxin. The redox potential difference between DsbA and thioredoxin has been measured to be 160 mV, equivalent to a shift of 15.4 kJ/mol in the reduced/oxidised equilibrium. Electrostatics calculations have been used to study the relative stabilities of the reduced forms of the two proteins. Model calculations suggest that much of the redox potential difference between DsbA and thioredoxin arises form altered stabilisation of the exposed and ionised thiolates of the reduced forms, supporting suggestions previously made on the basis of experimental studies. The calculations have been used to construct a molecular model for the difference in thiolate stabilisation. Although specific interactions, such as thiolate-NH 35 (thioredoxin)/33 (DsbA), provide substantial stabilisation in each reduced protein, the difference between thioredoxin and DsbA is predicted to reside in several side-chain and main-chain groups acting in concert. Residues H32 and Q97 in DsbA are predicted to contribute, along with substantial regions of the polypeptide backbone in the protein domain which is common to DsbA and thioredoxin. Increased thiolate stabilisation by the peptide dipoles is suggested to arise from altered main-chain disposition, and the effect of the additional protein domain of DsbA on the electric field. Peptide dipoles in a region of about 20 residues close to the active site disulphide are predicted to contribute significantly to the redox potential difference.

Recognition of novel and divergent higher plant chloroplast ribosomal proteins by Escherichia coli ribosome during in vivo assembly.

Architecture of higher plant chloroplast ribosomes involves additional protein domains over that found in the Escherichia coli ribosome, although the rRNAs in these two kinds of ribosomes are very similar in length and sequence (Subramanian, A. R. (1993) Trends Biochem. Sci. 18, 177-180). Here, we show that two chloroplast-specific protein domains (a novel chloroplast ribosomal protein of the 30 S subunit, called Psrp-1 or S22, and a divergent protein of the 50 S subunit with long terminal extensions and low homology to its E. coli counterpart, L21) are both incorporated in E. coli ribosomes and polysomes when their gene constructs are expressed in E. coli. Also, the 67-residue NH2-terminal extension in chloroplast L21 by itself is incorporated. Thus, our results indicate preexisting binding sites for novel chloroplast-specific ribosomal proteins/domains on eubacterial ribosomes. Additionally, we observed cleavage of the chloroplast-targeting transit peptide (present in the expressed Psrp-1 precursor), indicating protease(s) of the required specificity in E. coli cells. The expression of chloroplast L21 with its NH2-terminal extension was inhibitory to E. coli growth, suggesting a drastic effect of the latter on some property of L21. Expression of Psrp-1 was neutral, consistent with a function only in chloroplast translation. Based on analysis of the assembly of Psrp-1 and various L21 fragments in E. coli ribosomes, a general model for studying ribosomal protein-ribosome interactions is suggested.

An echistatin C-terminal peptide activates GPIIbIIIa binding to fibrinogen, fibronectin, vitronectin and collagen type I and type IV.

Integrin binding to proteins often involves recognition of domains containing the arginine-glycine-aspartate (RGD) motif. Different binding affinities and specificities of the integrin-ligand protein interactions involve additional protein domains. The n.m.r. structure of the snake-venom protein echistatin suggested that the C-terminal portion of the molecule might be important, in addition to the RGD domain, in binding to the integrin glycoprotein IIbIIIa (GPIIbIIIa) [Saudek, Atkinson and Pelton (1991) Biochem. 30, 7369-7372]. The synthetic C-terminal peptide, echistatin-(40-49), PRNPHKGPAT, (1) inhibited binding of GPIIbIIIa to immobilized echistatin (IC50 3-6 mM), but did not inhibit binding of GPIIbIIIa to immobilized fibrinogen (up to 5 mM peptide), (2) activated GPIIbIIIa binding to fibronectin and vitronectin, usual ligands for the activated integrin, (3) activated binding of GPIIbIIIa to collagen type I and type IV, proteins not usually regarded as ligands for the integrin, and (4) stimulated 125I-fibrinogen binding by human platelets. These findings argue for an interaction of this non-RGD domain in echistatin with GPIIbIIIa, leading to activation of the integrin and extension of the ligand specificity to include immobilized collagen.