In Vitro Studies with Purified Components Reveal Signal Recognition Particle (SRP) and SecA/SecB as Constituents of Two Independent Protein-targeting Pathways of Escherichia coli

The molecular requirements for the translocation of secretory proteins across, and the integration of membrane proteins into, the plasma membrane of Escherichia coli were compared. This was achieved in a novel cell-free system from E. coli which, by extensive subfractionation, was simultaneously rendered deficient in SecA/SecB and the signal recognition particle (SRP) components, Ffh (P48), 4.5S RNA, and FtsY. The integration of two membrane proteins into inside-out plasma membrane vesicles of E. coli required all three SRP components and could not be driven by SecA, SecB, and ΔμH+. In contrast, these were the only components required for the translocation of secretory proteins into membrane vesicles, a process in which the SRP components were completely inactive. Our results, while confirming previous in vivo studies, provide the first in vitro evidence for the dependence of the integration of polytopic inner membrane proteins on SRP in E. coli. Furthermore, they suggest that SRP and SecA/SecB have different substrate specificities resulting in two separate targeting mechanisms for membrane and secretory proteins in E. coli. Both targeting pathways intersect at the translocation pore because they are equally affected by a blocked translocation channel.

A dimeric crystal structure for the N-terminal two domains of intercellular adhesion molecule-1

The 3.0-Å structure of a 190-residue fragment of intercellular adhesion molecule-1 (ICAM-1, CD54) reveals two tandem Ig-superfamily (IgSF) domains. Each of two independent molecules dimerizes identically with a symmetry-related molecule over a hydrophobic interface on the BED sheet of domain 1, in agreement with dimerization of ICAM-1 on the cell surface. The residues that bind to the integrin LFA-1 are well oriented for bivalent binding in the dimer, with the critical Glu-34 residues pointing away from each other on the periphery. Residues that bind to rhinovirus are in the flexible BC and FG loops at the tip of domain 1, and these and the upper half of domain 1 are well exposed in the dimer for docking to virus. By contrast, a residue important for binding to Plasmodium falciparum-infected erythrocytes is in the dimer interface. The presence of A′ strands in both domains 1 and 2, conserved hydrogen bonds at domain junctions, and elaborate hydrogen bond networks around the key integrin binding residues in domain 1 make these domains suited to resist tensile forces during adhesive interactions. A subdivision of the intermediate (I) set of IgSF domains is proposed in which domain 1 of ICAM-1 and previously described I set domains belong to the I1 set and domain 2 of ICAM-1, ICAM-2, and vascular cell adhesion molecule-1 belong to the I2 set.

Mitosis in vertebrate somatic cells with two spindles: Implications for the metaphase/anaphase transition checkpoint and cleavage

During mitosis an inhibitory activity associated with unattached kinetochores prevents PtK1 cells from entering anaphase until all kinetochores become attached to the spindle. To gain a better understanding of how unattached kinetochores block the metaphase/anaphase transition we followed mitosis in PtK1 cells containing two independent spindles in a common cytoplasm. We found that unattached kinetochores on one spindle did not block anaphase onset in a neighboring mature metaphase spindle 20 μm away that lacked unattached kinetochores. As in cells containing a single spindle, anaphase onset occurred in the mature spindles x̄ = 24 min after the last kinetochore attached regardless of whether the adjacent immature spindle contained one or more unattached kinetochores. These findings reveal that the inhibitory activity associated with an unattached kinetochore is functionally limited to the vicinity of the spindle containing the unattached kinetochore. We also found that once a mature spindle entered anaphase the neighboring spindle also entered anaphase x̄ = 9 min later regardless of whether it contained monooriented chromosomes. Thus, anaphase onset in the mature spindle catalyzes a “start anaphase” reaction that spreads globally throughout the cytoplasm and overrides the inhibitory signal produced by unattached kinetochores in an adjacent spindle. Finally, we found that cleavage furrows often formed between the two independent spindles. This reveals that the presence of chromosomes and/or a spindle between two centrosomes is not a prerequisite for cleavage in vertebrate somatic cells.

Two terrestrial records of rapid climatic change during the glacial–Holocene transition (14,000– 9,000 calendar years B.P.) from Europe

Two independent multidisciplinary studies of climatic change during the glacial–Holocene transition (ca. 14,000–9,000 calendar yr B.P.) from Norway and Switzerland have assessed organism responses to the rapid climatic changes and made quantitative temperature reconstructions with modern calibration data sets (transfer functions). Chronology at Kråkenes, western Norway, was derived from calibration of a high-resolution series of 14C dates. Chronologies at Gerzensee and Leysin, Switzerland, were derived by comparison of δ18O in lake carbonates with the δ18O record from the Greenland Ice Core Project. Both studies demonstrate the sensitivity of terrestrial and aquatic organisms to rapid temperature changes and their value for quantitative reconstruction of the magnitudes and rates of the climatic changes. The rates in these two terrestrial records are comparable to those in Greenland ice cores, but the actual temperatures inferred apply to the terrestrial environments of the two regions.

Vps41p Function in the Alkaline Phosphatase Pathway Requires Homo-oligomerization and Interaction with AP-3 through Two Distinct Domains

Transport of proteins through the ALP (alkaline phosphatase) pathway to the vacuole requires the function of the AP-3 adaptor complex and Vps41p. However, unlike other adaptor protein–dependent pathways, the ALP pathway has not been shown to require additional accessory proteins or coat proteins, such as membrane recruitment factors or clathrin. Two independent genetic approaches have been used to identify new mutants that affect transport through the ALP pathway. These screens yielded new mutants in both VPS41 and the four AP-3 subunit genes. Two new VPS41 alleles exhibited phenotypes distinct from null mutants of VPS41, which are defective in vacuolar morphology and protein transport through both the ALP and CPY sorting pathways. The new alleles displayed severe ALP sorting defects, normal vacuolar morphology, and defects in ALP vesicle formation at the Golgi complex. Sequencing analysis of these VPS41 alleles revealed mutations encoding amino acid changes in two distinct domains of Vps41p: a conserved N-terminal domain and a C-terminal clathrin heavy-chain repeat (CHCR) domain. We demonstrate that the N-terminus of Vps41p is required for binding to AP-3, whereas the C-terminal CHCR domain directs homo-oligomerization of Vps41p. These data indicate that a homo-oligomeric form of Vps41p is required for the formation of ALP containing vesicles at the Golgi complex via interactions with AP-3.

A single-headed dimer of Escherichia coli ribosomal protein L7/L12 supports protein synthesis

During protein synthesis, the two elongation factors Tu and G alternately bind to the 50S ribosomal subunit at a site of which the protein L7/L12 is an essential component. L7/L12 is present in each 50S subunit in four copies organized as two dimers. Each dimer consists of distinct domains: a single N-terminal (“tail”) domain that is responsible for both dimerization and binding to the ribosome via interaction with the protein L10 and two independent globular C-terminal domains (“heads”) that are required for binding of elongation factors to ribosomes. The two heads are connected by flexible hinge sequences to the N-terminal domain. Important questions concerning the mechanism by which L7/L12 interacts with elongation factors are posed by us in response to the presence of two dimers, two heads per dimer, and their dynamic, mobile properties. In an attempt to answer these questions, we constructed a single-headed dimer of L7/L12 by using recombinant DNA techniques and chemical cross-linking. This chimeric molecule was added to inactive core particles lacking wild-type L7/L12 and shown to restore activity to a level approaching that of wild-type two-headed L7/L12.

Novel Ras antagonist blocks human melanoma growth

During past decades, knowledge of melanoma biology has increased considerably. Numerous therapeutic modalities based on this knowledge are currently under investigation. Advanced melanoma, nevertheless, remains a prime example of poor treatment response that may, in part, be the consequence of activated N-Ras oncoproteins. Besides oncogenic Ras, wild-type Ras gene products also play a key role in receptor tyrosine kinase growth factor signaling, known to be of importance in oncogenesis and tumor progression of a variety of human neoplasms, including malignant melanoma; therefore, it is reasonable to speculate that a pharmacological approach that curtails Ras activity may represent a sensible approach to inhibit melanoma growth. To test this concept, the antitumor activity of S-trans, trans-farnesylthiosalicylic acid (FTS), a recently discovered Ras antagonist that dislodges Ras from its membrane-anchoring sites, was evaluated. The antitumor activity of FTS was assessed both in vitro and in vivo in two independent SCID mouse xenotransplantation models of human melanoma expressing either wild-type Ras (cell line 518A2) or activated Ras (cell line 607B). We show that FTS (5–50 μM) reduces the amounts of activated N-Ras and wild-type Ras isoforms both in human melanoma cells and Rat-1 fibroblasts, interrupts the Ras-dependent extracellular signal-regulated kinase in melanoma cells, inhibits the growth of N-Ras-transformed fibroblasts and human melanoma cells in vitro and reverses their transformed phenotype. FTS also causes a profound and statistically significant inhibition of 518A2 (82%) and 607B (90%) human melanoma growth in SCID mice without evidence of drug-related toxicity. Our findings stress the notion that FTS may qualify as a novel and rational treatment approach for human melanoma and possibly other tumors that either carry activated ras genes or rely on Ras signal transduction more heavily than nonmalignant cells.

Ancient mtDNA sequences in the human nuclear genome: A potential source of errors in identifying pathogenic mutations

Nuclear-localized mtDNA pseudogenes might explain a recent report describing a heteroplasmic mtDNA molecule containing five linked missense mutations dispersed over the contiguous mtDNA CO1 and CO2 genes in Alzheimer’s disease (AD) patients. To test this hypothesis, we have used the PCR primers utilized in the original report to amplify CO1 and CO2 sequences from two independent ρ° (mtDNA-less) cell lines. CO1 and CO2 sequences amplified from both of the ρ° cells, demonstrating that these sequences are also present in the human nuclear DNA. The nuclear pseudogene CO1 and CO2 sequences were then tested for each of the five “AD” missense mutations by restriction endonuclease site variant assays. All five mutations were found in the nuclear CO1 and CO2 PCR products from ρ° cells, but none were found in the PCR products obtained from cells with normal mtDNA. Moreover, when the overlapping nuclear CO1 and CO2 PCR products were cloned and sequenced, all five missense mutations were found, as well as a linked synonymous mutation. Unlike the findings in the original report, an additional 32 base substitutions were found, including two in adjacent tRNAs and a two base pair deletion in the CO2 gene. Phylogenetic analysis of the nuclear CO1 and CO2 sequences revealed that they diverged from modern human mtDNAs early in hominid evolution about 770,000 years before present. These data would be consistent with the interpretation that the missense mutations proposed to cause AD may be the product of ancient mtDNA variants preserved as nuclear pseudogenes.

Cleavage Furrows Formed between Centrosomes Lacking an Intervening Spindle and Chromosomes Contain Microtubule Bundles, INCENP, and CHO1 but Not CENP-E

PtK1 cells containing two independent mitotic spindles can cleave between neighboring centrosomes, in the absence of an intervening spindle, as well as at the spindle equators. We used same-cell video, immunofluorescence, and electron microscopy to compare the structure and composition of normal equatorial furrows with that of ectopic furrows formed between spindles. As in controls, ectopic furrows contained midbodies composed of microtubule bundles and an electron-opaque matrix. Despite the absence of an intervening spindle and chromosomes, the midbodies associated with ectopic furrows also contained the microtubule-bundling protein CHO1 and the chromosomal passenger protein INCENP. However, CENP-E, another passenger protein, was not found in ectopic furrows but was always present in controls. We also examined cells in which the ectopic furrow initiated but relaxed. Although relaxing furrows contained overlapping microtubules from opposing centrosomes, they lacked microtubule bundles as well as INCENP and CHO1. Together these data suggest that the mechanism defining the site of furrow formation during mitosis in vertebrates does not depend on the presence of underlying microtubule bundles and chromosomes or on the stable association of INCENP or CHO1. The data also suggest that the completion of cytokinesis requires the presence of microtubule bundles and specific proteins (e.g., INCENP, CHO1, etc.) that do not include CENP-E.

Rab11 Is Required for Trans-Golgi Network–to–Plasma Membrane Transport and a Preferential Target for GDP Dissociation Inhibitor

The rab11 GTPase has been localized to both the Golgi and recycling endosomes; however, its Golgi-associated function has remained obscure. In this study, rab11 function in exocytic transport was analyzed by using two independent means to perturb its activity. First, expression of the dominant interfering rab11S25N mutant protein led to a significant inhibition of the cell surface transport of vesicular stomatitis virus (VSV) G protein and caused VSV G protein to accumulate in the Golgi. On the other hand, the expression of wild-type rab11 or the activating rab11Q70L mutant had no adverse effect on VSV G transport. Next, the membrane association of rab11, which is crucial for its function, was perturbed by modest increases in GDP dissociation inhibitor (GDI) levels. This led to selective inhibition of the trans-Golgi network to cell surface delivery, whereas endoplasmic reticulum–to–Golgi and intra-Golgi transport were largely unaffected. The transport inhibition was reversed specifically by coexpression of wild-type rab11 with GDI. Under the same conditions two other exocytic rab proteins, rab2 and rab8, remained membrane bound, and the transport steps regulated by these rab proteins were unaffected. Neither mutant rab11S25N nor GDI overexpression had any impact on the cell surface delivery of influenza hemagglutinin. These data show that functional rab11 is critical for the export of a basolateral marker but not an apical marker from the trans-Golgi network and pinpoint rab11 as a sensitive target for inhibition by excess GDI.

Coordinate Regulation of G- and C Strand Length during New Telomere Synthesis

We have used the ciliate Euplotes to study the role of DNA polymerase in telomeric C strand synthesis. Euplotes provides a unique opportunity to study C strand synthesis without the complication of simultaneous DNA replication because millions of new telomeres are made at a stage in the life cycle when no general DNA replication takes place. Previously we showed that the C-strands of newly synthesized telomeres have a precisely controlled length while the G-strands are more heterogeneous. This finding suggested that, although synthesis of the G-strand (by telomerase) is the first step in telomere addition, a major regulatory step occurs during subsequent C strand synthesis. We have now examined whether G- and C strand synthesis might be regulated coordinately rather than by two independent mechanisms. We accomplished this by determining what happens to G- and C strand length if C strand synthesis is partially inhibited by aphidicolin. Aphidicolin treatment caused a general lengthening of the G-strands and a large increase in C strand heterogeneity. This concomitant change in both the G- and C strand length indicates that synthesis of the two strands is coordinated. Since aphidicolin is a very specific inhibitor of DNA polα and polδ, our results suggest that this coordinate length regulation is mediated by DNA polymerase.

Class VI Unconventional Myosin is Required for Spermatogenesis in Drosophila

We have identified partial loss of function mutations in class VI unconventional myosin, 95F myosin, which results in male sterility. During spermatogenesis the germ line precursor cells undergo mitosis and meiosis to form a bundle of 64 spermatids. The spermatids remain interconnected by cytoplasmic bridges until individualization. The process of individualization involves the formation of a complex of cytoskeletal proteins and membrane, the individualization complex (IC), around the spermatid nuclei. This complex traverses the length of each spermatid resolving the shared membrane into a single membrane enclosing each spermatid. We have determined that 95F myosin is a component of the IC whose function is essential for individualization. In wild-type testes, 95F myosin localizes to the leading edge of the IC. Two independent mutations in 95F myosin reduce the amount of 95F myosin in only a subset of tissues, including the testes. This reduction of 95F myosin causes male sterility as a result of defects in spermatid individualization. Germ line transformation with the 95F myosin heavy chain cDNA rescues the male sterility phenotype. IC movement is aberrant in these 95F myosin mutants, indicating a critical role for 95F myosin in IC movement. This report is the first identification of a component of the IC other than actin. We propose that 95F myosin is a motor that participates in membrane reorganization during individualization.

No ·NO from NO synthase

The nitric-oxide synthase (NOS; EC 1.14.13.39) reaction is formulated as a partially tetrahydrobiopterin (H4Bip)-dependent 5-electron oxidation of a terminal guanidino nitrogen of l-arginine (Arg) associated with stoichiometric consumption of dioxygen (O2) and 1.5 mol of NADPH to form l-citrulline (Cit) and nitric oxide (·NO). Analysis of NOS activity has relied largely on indirect methods such as quantification of nitrite/nitrate or the coproduct Cit; we therefore sought to directly quantify ·NO formation from purified NOS. However, by two independent methods, NOS did not yield detectable ·NO unless superoxide dismutase (SOD; EC 1.15.1.1) was present. In the presence of H4Bip, internal ·NO standards were only partially recovered and the dismutation of superoxide (O2⨪), which otherwise scavenges ·NO to yield ONOO−, was a plausible mechanism of action of SOD. Under these conditions, a reaction between NADPH and ONOO− resulted in considerable overestimation of enzymatic NADPH consumption. SOD lowered the NADPH:Cit stoichiometry to 0.8–1.1, suggesting either that additional reducing equivalents besides NADPH are required to explain Arg oxidation to ·NO or that ·NO was not primarily formed. The latter was supported by an additional set of experiments in the absence of H4Bip. Here, recovery of internal ·NO standards was unaffected. Thus, a second activity of SOD, the conversion of nitroxyl (NO−) to ·NO, was a more likely mechanism of action of SOD. Detection of NOS-derived nitrous oxide (N2O) and hydroxylamine (NH2OH), which cannot arise from ·NO decomposition, was consistent with formation of an ·NO precursor molecule such as NO−. When, in the presence of SOD, glutathione was added, S-nitrosoglutathione was detected. Our results indicate that ·NO is not the primary reaction product of NOS-catalyzed Arg turnover and an alternative reaction mechanism and stoichiometry have to be taken into account.

Polynesian origins: Insights from the Y chromosome

The question surrounding the colonization of Polynesia has remained controversial. Two hypotheses, one postulating Taiwan as the putative homeland and the other asserting a Melanesian origin of the Polynesian people, have received considerable attention. In this work, we present haplotype data based on the distribution of 19 biallelic polymorphisms on the Y chromosome in a sample of 551 male individuals from 36 populations living in Southeast Asia, Taiwan, Micronesia, Melanesia, and Polynesia. Surprisingly, nearly none of the Taiwanese Y haplotypes were found in Micronesia and Polynesia. Likewise, a Melanesian-specific haplotype was not found among the Polynesians. However, all of the Polynesian, Micronesian, and Taiwanese haplotypes are present in the extant Southeast Asian populations. Evidently, the Y-chromosome data do not lend support to either of the prevailing hypotheses. Rather, we postulate that Southeast Asia provided a genetic source for two independent migrations, one toward Taiwan and the other toward Polynesia through island Southeast Asia.

How the owl resolves auditory coding ambiguity

The barn owl (Tyto alba) uses interaural time difference (ITD) cues to localize sounds in the horizontal plane. Low-order binaural auditory neurons with sharp frequency tuning act as narrow-band coincidence detectors; such neurons respond equally well to sounds with a particular ITD and its phase equivalents and are said to be phase ambiguous. Higher-order neurons with broad frequency tuning are unambiguously selective for single ITDs in response to broad-band sounds and show little or no response to phase equivalents. Selectivity for single ITDs is thought to arise from the convergence of parallel, narrow-band frequency channels that originate in the cochlea. ITD tuning to variable bandwidth stimuli was measured in higher-order neurons of the owl’s inferior colliculus to examine the rules that govern the relationship between frequency channel convergence and the resolution of phase ambiguity. Ambiguity decreased as stimulus bandwidth increased, reaching a minimum at 2–3 kHz. Two independent mechanisms appear to contribute to the elimination of ambiguity: one suppressive and one facilitative. The integration of information carried by parallel, distributed processing channels is a common theme of sensory processing that spans both modality and species boundaries. The principles underlying the resolution of phase ambiguity and frequency channel convergence in the owl may have implications for other sensory systems, such as electrolocation in electric fish and the computation of binocular disparity in the avian and mammalian visual systems.