A region of group I introns that contains universally conserved residues but is not essential for self-splicing.

The catalytic core of the self-splicing group I intron RNAs is composed of six paired regions together with their connecting sequences; these are thought to form two elongated domains, with paired regions P5, P4, and P6 aligned along one axis and P8, P3, and P7 along the other. Most of the very highly conserved residues of the group I introns lie in or near P7, but two occur in L4, the internal loop connecting P4 and P5. It is generally believed that such bases are conserved because they are essential for splicing. Mutants were created in a member of each of the two major subclasses of group I introns, in which P5, L4, and the distal portion of P4 were deleted. Splicing activity was still detected in these mutants, albeit substantially weakened; splicing was accurate and occurred by the normal group I mechanism, with addition of a guanosine molecule to the intron. Thus the deleted region, containing two universally conserved bases, is not essential but facilitates splicing. Another reaction characteristic of group I introns, hydrolysis of the 3' splice site, was less severely affected by the deletions. The results are discussed in terms of the prevailing three-dimensional model for the core structure of the group I introns.     

Identification of a Steap3 endosomal targeting motif essential for normal iron metabolism

Hereditary forms of iron-deficiency anemia, including animal models, have taught us much about the normal physiologic control of iron metabolism. However, the discovery of new informative mutants is limited by the natural mutation frequency. To address this limitation, we have developed a screen for heritable abnormalities of red blood cell morphology in mice with single-nucleotide changes induced by the chemical mutagen ethylnitrosourea (ENU). We now describe the first strain, fragile-red, with hypochromic microcytic anemia resulting from a Y228H substitution in the ferrireductase Steap3 (Steap3(Y288H)). Analysis of the Steap3(Y288H) mutant identifies a conserved motif required for targeting Steap3 to internal compartments and highlights how phenotypic screens linked to mutagenesis can identify new functional variants in erythropoiesis and ascribe function to previously unidentified motifs.     

The unstructured linker of Mlh1 contains a motif required for endonuclease function which is mutated in cancers

Eukaryotic DNA mismatch repair (MMR) depends on recruitment of the Mlh1-Pms1 endonuclease (human MLH1-PMS2) to mispaired DNA. Both Mlh1 and Pms1 contain a long unstructured linker that connects the N- and carboxyl-terminal domains. Here, we demonstrated the Mlh1 linker contains a conserved motif (Saccharomyces cerevisiae residues 391–415) required for MMR. The Mlh1-R401A,D403A-Pms1 linker motif mutant protein was defective for MMR and endonuclease activity in vitro, even though the conserved motif could be >750 Å from the carboxyl-terminal endonuclease active site or the N-terminal adenosine triphosphate (ATP)-binding site. Peptides encoding this motif inhibited wild-type Mlh1-Pms1 endonuclease activity. The motif functioned in vivo at different sites within the Mlh1 linker and within the Pms1 linker. Motif mutations in human cancers caused a loss-of-function phenotype when modeled in S. cerevisiae. These results suggest that the Mlh1 motif promotes the PCNA-activated endonuclease activity of Mlh1-Pms1 via interactions with DNA, PCNA, RFC, or other domains of the Mlh1-Pms1 complex.

DNA mismatch repair (MMR) acts on mispairs arising from DNA-replication errors, formation of homologous recombination intermediates, and some chemically modified DNA bases (1–3). During MMR, mispair recognition by MutS homologs, primarily Msh2-Msh6 and Msh2-Msh3 in eukaryotes (4–8), is required to recruit MutL homologs to mispaired DNA, primarily Mlh1-Pms1 in eukaryotes (called MLH1-PMS2 in humans) (1–3, 9). In organisms other than Escherichia coli and related bacteria (10), the MutL homologs have an endonuclease activity that specifically nicks double-stranded DNA on strands containing pre-existing nicks (11–13). Nicking by Mlh1-Pms1 in vitro is required for Exo1-mediated repair on substrates with a nick 3′ to the mispair, as formation of a strand-specific nick 5′ to the mispair allows the 5′–3′ exonuclease activity of Exo1 to excise the mispair (11–14). The absolute requirement of this Mlh1-Pms1 nicking activity in vivo is not well understood, as both 5′ and 3′ nicks relative to mispairs are likely already present on newly synthesized DNA strands (15, 16). One proposal suggests that Mlh1-Pms1 activity maintains single-stranded discontinuities, which appear to identify the newly synthesized strand, even in the presence of the competing activities, like DNA ligation and gap filling by DNA polymerases (15, 17).MutL homologs are comprised of an N-terminal GHKL family adenosine triphosphatase (ATPase) domain, a carboxyl-terminal dimerization domain, and a predicted unstructured linker domain that connects the folded N- and carboxyl-terminal domains (18–21). In Saccharomyces cerevisiae, the unstructured linkers of Mlh1 and Pms1 are ∼150 and 250 amino acids long, respectively (22). These linkers have a biased sequence composition with reduced hydrophobic amino acids, like the large (>50 amino acid) intrinsically disordered regions (IDRs) present in many proteins (23–25). IDRs often mediate intermolecular interactions, play functional roles, and sometimes become ordered when bound to partners (23–25).MutL homologs, including Mlh1-Pms1, form DNA-bound rings called sliding clamps following loading by MutS homologs, ATP binding, and dimerization of the N-terminal ATPase domains; these rings rapidly diffuse along the DNA axis (26–30). The extended length of the unstructured interdomain linkers has been suggested to allow these MutL homolog clamps to migrate past protein–DNA complexes, which are normally a barrier to MutS homolog clamps, although Msh2-Msh3 clamps appear to be able to open and close on encountering a protein–DNA complex and hop over it (26–29, 31, 32). Remarkably, cleavage of the S. cerevisiae Mlh1 linker in vivo causes increased mutation rates, suggesting that intact sliding clamps are important for MMR (22). The importance of the combined lengths of the Mlh1 and Pms1 linkers in vivo is suggested by the synergistic increases in mutation rate that have been observed when combining S. cerevisiae mlh1 and pms1 mutations that shorten the linkers (26). In contrast, some linker missense mutations, which do not alter linker lengths, cause MMR defects (22, 33–35). Moreover, deletions within the S. cerevisiae Mlh1 linker tend to cause MMR defects, whereas deletions in the S. cerevisiae Pms1 linker tend not to, except for the pms1-Δ390–610 deletion that eliminates almost the entire Pms1 linker, resulting in a mutant complex that cannot be recruited by Msh2–Msh6 to mispair-containing DNA and fails to bind to DNA under low ionic strength conditions (22). Together, the data suggest that length is only one requirement for the Mlh1 and Pms1 linkers and that the Mlh1 and Pms1 linkers differ in importance for MMR.Here, we have identified a motif in the Mlh1 linker, which spans residues 391–415, that is conserved from S. cerevisiae to humans and is required for MMR. Mutation of two of the residues in this motif, R401 and I409, to alanine caused an MMR defect, as did short deletions affecting other partially conserved residues within the motif. We found that the motif was functional when moved to different positions on the Mlh1 linker and when the distances between motif and the folded N- and carboxyl-terminal domains were altered. Moreover, moving a copy of the motif to the Pms1 subunit complemented the MMR defect caused by loss of the motif in Mlh1; in addition, swapping the Mlh1 linker with the Pms1 linker supported MMR. Mutant Mlh1-Pms1 complexes with amino acid substitutions in the conserved Mlh1 motif could not support reconstituted MMR reactions in vitro and were defective for Mlh1-Pms1 endonuclease activity but were recruited to mispair-containing DNA by Msh2-Msh6. Peptides encoding the conserved motif, but not control peptides, inhibited wild-type Mlh1-Pms1 endonuclease activity. Consistent with these observations, increased levels of Pms1-4GFP foci, which are MMR intermediates (36), were caused by mutations disrupting the conserved Mlh1 motif, similar to other mutations that reduce Mlh1-Pms1 endonuclease activity (36–38). Mutations of the motif were observed in human cancers, and these mutations disrupted MMR in vivo when modeled in the S. cerevisiae MLH1 gene. Taken together, these data are consistent with a requirement of the Mlh1 linker motif for Mlh1-Pms1 endonuclease activity in MMR, which could be due to an interaction of the motif with the DNA substrate, with the endonuclease active site, and/or with the endonuclease-activating PCNA.     

The 3'' untranslated region of localized maternal messages contains a conserved motif involved in mRNA localization.

Messenger RNA (mRNA) localization is emerging as a means of regulating gene expression. This process is operational in fly and frog development, where a subset of maternally inherited RNAs are asymmetrically distributed and thought to impart axial polarity to the embryo. Since most maternal mRNAs are uniformly distributed, an apparatus must exist to recognize and specifically transport these rare localized species. Here I report the identification of a nine-nucleotide motif, YUGUUYCUG, common to the 3' untranslated regions of four sequenced messages of this class: Drosophila bicoid and nanos mRNAs and Xenopus An2 and Vg1 mRNAs. To test the role of this nonamer sequence in the localization process, a Drosophila transient assay has been established. The assay reveals that bicoid mRNA specifically lacking this nonamer is partially mislocalized. In contrast, nonamer deletion is inconsequential to message stability. The existence of specific and general mRNA localization signals is proposed and it is suggested that this conserved motif belongs to the latter category.     

Delineation of a region in the B2 bradykinin receptor that is essential for high-affinity agonist binding.

We have made mutations in the predicted sixth transmembrane segment of a rat B2 bradykinin receptor and analyzed the variant proteins by expressing them in COS-1 cells. Two amino acid substitutions reduced the affinity of the receptor for bradykinin (Phe261-->Val by 1600-fold; Thr265-->Ala by 700-fold) with comparatively little effect on the affinity for the bradykinin antagonists NPC17731 and D-Arg-[Hyp3,D-Phe7]bradykinin (where Hyp is hydroxyproline). Three other substitutions (Gln262-->Ala, Asp268-->Ala, and Thr269-->Ala) modestly reduced the affinity for bradykinin and for the antagonist D-Arg-[Hyp3,D-Phe7]bradykinin. Even the most dramatically affected mutated receptors were still able to couple, after bradykinin binding, to phosphatidylinositol turnover. The data suggest that bradykinin directly contacts the face of the sixth transmembrane helix formed by the residues Phe261, Gln262, Thr265, Asp268, and Thr269 or that this face of the helix is the site of intraprotein contacts that serve to stabilize the agonist-binding conformation of the receptor.     

An intact PDZ motif is essential for correct P2Y12 purinoceptor traffic in human platelets

The platelet P2Y(12) purinoceptor (P2Y(12)R), which plays a crucial role in hemostasis, undergoes internalization and subsequent recycling to maintain receptor responsiveness, processes that are essential for normal platelet function. Here, we observe that P2Y(12)R function is compromised after deletion or mutation of the 4 amino acids at the extreme C-terminus of this receptor (ETPM), a putative postsynaptic density 95/disc large/zonula occludens-1 (PDZ)-binding motif. In cell line models, removal of this sequence or mutation of one of its core residues (P341A), attenuates receptor internalization and receptor recycling back to the membrane, thereby blocking receptor resensitization. The physiologic significance of these findings in the regulation of platelet function is shown by identification of a patient with a heterozygous mutation in the PDZ binding sequence of their P2Y(12)R (P341A) that is associated with reduced expression of the P2Y(12)R on the cell surface. Importantly, platelets from this subject showed significantly compromised P2Y(12)R recycling, emphasizing the importance of the extreme C-terminus of this receptor to ensure correct receptor traffic.     

The CDC7 protein of Saccharomyces cerevisiae is a phosphoprotein that contains protein kinase activity.

The CDC7 protein of Saccharomyces cerevisiae may be involved in the G1/S-phase transition and/or in the initiation of mitotic DNA synthesis. The CDC7 gene has two in-frame AUG codons as possible translation start sites, which would produce 58- and 56-kDa proteins, respectively. Both p58 and p56 derived from recombinant plasmids complement the temperature-sensitive growth defect of the cdc7-1 allele. To determine the biochemical function of the CDC7 protein, the CDC7 gene was cloned and polyclonal antibodies were produced against the CDC7 protein. CDC7 immune complexes prepared from yeast with these antibodies phosphorylate histone H1. Kinase activity is thermolabile in strains carrying the cdc7-1 temperature-sensitive mutant allele and is elevated greater than 10-fold in strains carrying plasmids overexpressing either p56 or p58, confirming that the kinase in the immunoprecipitates is the CDC7 gene product. In addition, we show that CDC7 is a phosphoprotein itself. Indirect immunofluorescence and biochemical fractionation show that the CDC7 protein is present at relatively high concentrations in the nucleus compared with the cytoplasm, suggesting that nuclear proteins may be substrates for the CDC7 protein.     

Pseudomonas exotoxin contains a specific sequence at the carboxyl terminus that is required for cytotoxicity.

Pseudomonas exotoxin (PE), a single-chain polypeptide toxin of 613 amino acids, consists of three functional domains: an amino-terminal receptor-binding domain, a middle translocation domain, and a carboxyl-terminal ADP-ribosylation domain. Deletion of as few as 2 or as many as 11 amino acids from the carboxyl terminus of PE does not affect ADP-ribosylation activity but produces noncytotoxic molecules. Deletions and substitutions between positions 602 and 611 of PE show that the last 5 amino acids of PE are very important for its cytotoxic action. The carboxyl-terminal sequence of PE is Arg-Glu-Asp-Leu-Lys. Mutational analysis indicates that a basic amino acid at 609, acidic amino acids at 610 and 611, and a leucine at 612 are required for full cytotoxic activity. Lysine at 613 can be deleted or replaced with arginine but not with several other amino acids. Mutant toxins are able to bind normally to target Swiss mouse 3T3 cells and are internalized by endocytosis, but apparently they do not penetrate into the cytosol. A PE molecule that ends with Lys-Asp-Glu-Leu, which is a well defined endoplasmic reticulum retention sequence [Munro, S. and Pelham, R. B. (1987) Cell 48, 899-907], is fully cytotoxic, suggesting that a common factor may be involved in intoxication of cells by PE and retention of proteins in the lumen of the endoplasmic reticulum. Sequences similar to those at the carboxyl end of PE are also found at the end of Cholera toxin A chain and Escherichia coli heat-labile toxin A chain.     

Biosynthesis of granule proteins in normal human bone marrow cells. Gelatinase is a marker of terminal neutrophil differentiation

Differentiation and maturation of myeloid cells is characterized by the sequential acquisition of two distinct cytoplasmic granule subsets, azurophil granules and specific granules. We recently showed the existence of a third granule subset, gelatinase granules. To investigate whether appearance of gelatinase granules marks a further step in maturation of myeloid cells beyond the appearance of specific granules, we sorted normal human bone marrow cells into one of three groups according to maturity by centrifugation on Percoll density gradients. The biosynthesis of myeloperoxidase (MPO) (an azurophil granule marker), lactoferrin and neutrophil gelatinase-associated lipocalin NGAL (specific granules markers) and gelatinase was then studied in each of these groups. We found that gelatinase was synthesized mainly in the group containing band cells and segmented cells. This contrasted with lactoferrin and NGAL, which were synthesized almost exclusively in the group containing myelocytes and metamyelocytes, and with MPO, which was mainly synthesized in the group containing myeloblasts and promyelocytes. Immunocytochemistry was in full agreement with the biosynthesis data, and showed that gelatinase appears in band cells, whereas NGAL and lactoferrin both appear in myelocytes. Thus, acquisition of gelatinase granules marks a step in neutrophil differentiation beyond the appearance of specific granules.     

The PDZ-binding motif of the beta2-adrenoceptor is essential for physiologic signaling and trafficking in cardiac myocytes

beta1- and beta2-adrenergic receptors (AR) regulate cardiac myocyte function through distinct signaling pathways. In addition to regulating cardiac rate and contractility, beta1AR and beta2AR may play different roles in the pathogenesis of heart failure. Studies on neonatal cardiac myocytes from beta1AR and beta2AR knockout mice suggest that subtype-specific signaling is determined by subtype-specific membrane targeting and trafficking. Stimulation of beta2ARs has a biphasic effect on contraction rate, with an initial increase followed by a sustained Gi-dependent decrease. Recent studies show that a PDZ domain-binding motif at the carboxyl terminus of human beta2AR interacts with ezrin-binding protein 50/sodium-hydrogen exchanger regulatory factor, a PDZ-domain-containing protein. The human beta2AR carboxyl terminus also binds to N-ethylmaleimide-sensitive factor, which does not contain a PDZ domain. We found that mutation of the three carboxyl-terminal amino acids in the mouse beta2AR (beta2AR-AAA) disrupts recycling of the receptor after agonist-induced internalization in cardiac myocytes. Nevertheless, stimulation of the beta2AR-AAA produced a greater contraction rate increase than that of the wild-type beta2AR. This enhanced stimulation of contraction rate can be attributed in part to the failure of the beta2AR-AAA to couple to Gi. We also observed that coupling of endogenous, wild-type beta2AR to Gi in beta1AR knockout myocytes is inhibited by treatment with a membrane-permeable peptide representing the beta2AR carboxyl terminus. These studies demonstrate that association of the carboxyl terminus of the beta2AR with ezrin-binding protein 50/sodium-hydrogen exchanger regulatory factor, N-ethylmaleimide-sensitive factor, or some related proteins dictates physiologic signaling specificity and trafficking in cardiac myocytes.     

The cultural niche: why social learning is essential for human adaptation

In the last 60,000 y humans have expanded across the globe and now occupy a wider range than any other terrestrial species. Our ability to successfully adapt to such a diverse range of habitats is often explained in terms of our cognitive ability. Humans have relatively bigger brains and more computing power than other animals, and this allows us to figure out how to live in a wide range of environments. Here we argue that humans may be smarter than other creatures, but none of us is nearly smart enough to acquire all of the information necessary to survive in any single habitat. In even the simplest foraging societies, people depend on a vast array of tools, detailed bodies of local knowledge, and complex social arrangements and often do not understand why these tools, beliefs, and behaviors are adaptive. We owe our success to our uniquely developed ability to learn from others. This capacity enables humans to gradually accumulate information across generations and develop well-adapted tools, beliefs, and practices that are too complex for any single individual to invent during their lifetime.     

Evidence that subcellular localization of a bacterial membrane protein is achieved by diffusion and capture

Bacteria lack an endoplasmic reticulum, a Golgi apparatus, and transport vesicles and yet are capable of sorting and delivering integral membrane proteins to particular sites within the cell with high precision. What is the pathway by which membrane proteins reach their proper subcellular destination in bacteria? We have addressed this question by using green fluorescent protein (GFP) fused to a polytopic membrane protein (SpoIVFB) that is involved in the process of sporulation in the bacterium Bacillus subtilis. SpoIVFB-GFP localizes to a region of the sporulating cell known as the outer forespore membrane, which is distinct from the cytoplasmic membrane. Experiments are presented that rule out a mechanism in which SpoIVFB-GFP localizes to all membranes but is selectively eliminated from the cytoplasmic membrane by proteolytic degradation and argue against a model in which SpoIVFB-GFP is selectively inserted into the outer forespore membrane. Instead, the results are most easily compatible with a model in which SpoIVFB-GFP achieves proper localization by insertion into the cytoplasmic membrane followed by diffusion to, and capture in, the outer forespore membrane. The possibility that diffusion and capture is a general feature of protein localization in bacteria is discussed.