Molecular mechanism of cargo recognition and handover by the mammalian signal recognition particle.

Co-translational protein targeting to membranes by the signal recognition particle (SRP) is a universally conserved pathway from bacteria to humans. In mammals, SRP and its receptor (SR) have many additional RNA features and protein components compared to the bacterial system, which were recently shown to play regulatory roles. Due to its complexity, the mammalian SRP targeting process is mechanistically not well understood. In particular, it is not clear how SRP recognizes translating ribosomes with exposed signal sequences and how the GTPase activity of SRP and SR is regulated. Here, we present electron cryo-microscopy structures of SRP and SRP·SR in complex with the translating ribosome. The structures reveal the specific molecular interactions between SRP and the emerging signal sequence and the elements that regulate GTPase activity of SRP·SR. Our results suggest the molecular mechanism of how eukaryote-specific elements regulate the early and late stages of SRP-dependent protein targeting.

Structural Basis of Teneurin-Latrophilin Interaction in Repulsive Guidance of Migrating Neurons.

Teneurins are ancient metazoan cell adhesion receptors that control brain development and neuronal wiring in higher animals. The extracellular C terminus binds the adhesion GPCR Latrophilin, forming a trans-cellular complex with synaptogenic functions. However, Teneurins, Latrophilins, and FLRT proteins are also expressed during murine cortical cell migration at earlier developmental stages. Here, we present crystal structures of Teneurin-Latrophilin complexes that reveal how the lectin and olfactomedin domains of Latrophilin bind across a spiraling beta-barrel domain of Teneurin, the YD shell. We couple structure-based protein engineering to biophysical analysis, cell migration assays, and in utero electroporation experiments to probe the importance of the interaction in cortical neuron migration. We show that binding of Latrophilins to Teneurins and FLRTs directs the migration of neurons using a contact repulsion-dependent mechanism. The effect is observed with cell bodies and small neurites rather than their processes. The results exemplify how a structure-encoded synaptogenic protein complex is also used for repulsive cell guidance.

Structure of a prehandover mammalian ribosomal SRP·SRP receptor targeting complex.

Signal recognition particle (SRP) targets proteins to the endoplasmic reticulum (ER). SRP recognizes the ribosome synthesizing a signal sequence and delivers it to the SRP receptor (SR) on the ER membrane followed by the transfer of the signal sequence to the translocon. Here, we present the cryo-electron microscopy structure of the mammalian translating ribosome in complex with SRP and SR in a conformation preceding signal sequence handover. The structure visualizes all eukaryotic-specific SRP and SR proteins and reveals their roles in stabilizing this conformation by forming a large protein assembly at the distal site of SRP RNA. We provide biochemical evidence that the guanosine triphosphate hydrolysis of SRP·SR is delayed at this stage, possibly to provide a time window for signal sequence handover to the translocon.

Single copies of Sec61 and TRAP associate with a nontranslating mammalian ribosome.

During cotranslational protein translocation, the ribosome associates with a membrane channel, formed by the Sec61 complex, and recruits the translocon-associated protein complex (TRAP). Here we report the structure of a ribosome-channel complex from mammalian endoplasmic reticulum in which the channel has been visualized at 11 A resolution. In this complex, single copies of Sec61 and TRAP associate with a nontranslating ribosome and this stoichiometry was verified by quantitative mass spectrometry. A bilayer-like density surrounds the channel and can be attributed to lipid and detergent. The crystal structure of an archaeal homolog of the Sec61 complex was then docked into the map. In this model, two cytoplasmic loops of Sec61 may interact with RNA helices H6, H7, and H50, while the central pore is located below the ribosome tunnel exit. Hence, this copy of Sec61 is positioned to capture and translocate the nascent chain. Finally, we show that mammalian and bacterial ribosome-channel complexes have similar architectures.

Binding of nanoparticle receptors to peptide alpha-helices using amino acid-functionalized nanoparticles.

Nanoparticles provide large surface areas and controlled surface functionality and structure, making them excellent scaffolds for peptide recognition. A family of nanoparticles has been fabricated by amino acid functionalization to afford tailored surfaces. These particles are complementary to a tetraaspartate peptide (TAP) featuring cofacial anionic functionality when in the alpha-helical conformation. The functional groups present on these nanoparticle surfaces provide a tool to investigate the contribution of various noncovalent interactions at the nanoparticle-peptide interface. The ability of these particles to enforce the folding of the peptide into an alpha-helix was explored, demonstrating high helicity induction with particles featuring dicationic amino acids such as lysine or histidine, and little or no helix stabilization with hydrophobic amino acid termini.

Alpha-fetoprotein binding and uptake by primary cultures of human skeletal muscle.

alpha-Fetoprotein (AFP), a serum alpha-globulin mainly synthesized by the fetal liver and the yolk sac, is the major carrier of polyunsaturated fatty acids during embryo-fetal development. One property characteristic of fetal cells undergoing growth and differentiation is their ability to bind and internalize AFP. In the present work, we have studied the binding and endocytosis of AFP by human muscular cells developing in vitro. Primary cultures of human skeletal muscle, obtained from biopsies and examined at two stages of differentiation (myoblasts and myotubes), were incubated for different times, at 0 and 37 degrees C, with a colloidal-gold-conjugated human AFP probe and studied by light and electron microscopy, as well as by laser scanning confocal microscopy in the reflection mode. The results obtained show that (a) human myoblasts in primary culture bind and internalize the protein, probably through specific AFP receptors, (b) this property is strongly reduced or lost in well-differentiated myotubes, and (c) AFP is also bound, throughout culture development, to the extracellular matrix of fusing myoblasts and differentiated myotubes. The physiological significance of AFP uptake by human myoblasts undergoing growth and differentiation may be based on the ability of AFP to carry and deliver fatty acids to fetal cells.

[A morphofunctional analysis of the nuclear apparatus of the nonapeptidergic neurosecretory cells in the vertebrate hypothalamus]. / Morfofunktsional'nyi analiz iadernogo apparata nonapeptidergicheskikh neirosekretornykh kletok gipotalamusa pozvonochnykh.

A vast comparative material obtained on fishes, amphibians and mammals has been analysed using ecological and experimental histophysiological approaches. The structural organization of the nuclear apparatus of nonapeptidergic neurosecretory cells, especially in the lower vertebrates has been clearly shown to differ from that of the classic neurones. Morphometrical characteristics of nonapeptidergic neurosecretory cells of hypothalamic centers can be used for a reliable evaluation of their functional conditions. A high intensity of protein metabolism in nonapeptidergic cells of magnocellular hypothalamic centers leads to a bigger nucleolus/nucleus relation for these cells in parvocellular centers. All this proves the necessity of the morpho-functional analysis of nuclear apparatus of neurosecretory cells.

Cytosolic factors block antibody binding to the C-terminal cytoplasmic tail of the KDEL receptor.

The mammalian KDEL receptor is an extremely hydrophobic membrane protein. One of the longest stretches of hydrophilic sequence resides at the C-terminus. Various antibodies against a synthetic peptide corresponding to this region confirmed that the C-terminus is exposed to the cytoplasm. It was observed that antibody binding to the C-terminus of the KDEL receptor was diminished during immunofluorescence microscopy procedures which involved fixation prior to permeabilization as compared to when cells were permeabilized before fixation. Binding of both polyclonal and monoclonal antibodies, as assessed by indirect immunofluorescence microscopy in digitonin permeabilized cells, was inhibited by preincubation with rat liver cytosol. This inhibition was not observed with antibody against another membrane protein (p28) with a cytoplasmically exposed epitope also residing in the Golgi/intermediate compartment. Rabbit reticulocyte lysate had a similar effect while Schizosaccharomyces pombe cytosol inhibited binding to a greater degree than Saccharomyces cerevisiae cytosol. This inhibition by cytosol was prevented by coincubation with the antibody and was dose-dependent on the cytosol. Inhibition did not occur on ice or at 15 degrees C, or when the cytosol was energy-depleted by apyrase treatment. Interestingly, pretreatment of permeabilized cells with N-ethylmaleimide or its addition into the incubation mixture abolished inhibition. N-ethylmaleimide-treated cytosol, however, remained inhibitory. The findings suggest the existence of cytosolic factor (s) which interacts specifically with the cytoplasmic C-terminus of the KDEL receptor, which are likely to be components of the KDEL protein retrieval machinery.