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Cell recognition molecules of the immunoglobulin superfamily are involved in the formation, establishment, and plasticity of neural circuits in the central nervous system (CNS). We used a polymerase chain reaction-based approach to specifically amplify molecules with conserved sequence elements of immunoglobulin-like domains. This approach enabled us to isolate Kilon, a novel immunoglobulin that has been described by Funatsu et al. (J Biol Chem 1999;274:8224–8230) from the hippocampus. The sequence of Kilon shows a high degree of homology to that of the chicken protein neurotractin, a molecule involved in neurite outgrowth and capable of interacting with LAMP. In situ hybridization analysis was performed to analyze the Kilon mRNA distribution in the developing and adult rat brain and to compare it to that of LAMP mRNA. Kilon mRNA was found to be specifically expressed in the dentate gyrus (DG) of the adult rat, whereas LAMP transcripts were present in all regions of the hippocampal formation. These results were corroborated by RT-PCR semiquantification of gene expression in microdissected tissue prepared from the DG and the CA1 region of the hippocampus. We also performed mRNA expression analysis of both genes following hippocampal deafferentation and seizure, but neither Kilon nor LAMP gene expression showed significant alterations after lesioning on the in situ hybridization level. Our results show that the expression patterns of Kilon and LAMP during development and in the mature hippocampus are clearly distinguishable from one another, which suggests different roles for these related molecules in the hippocampus. Hippocampus 2000;10:632–644. © 2000 Wiley-Liss, Inc.

Protein translocation across the membrane of the endoplasmic reticulum (ER) proceeds through a proteinaceous translocation machinery, the translocon. To identify components that may regulate translocation by interacting with nascent polypeptides in the translocon, we used site-specific photo-crosslinking. We found that a region C-terminal of the two N-glycosylation sites of the MHC class II-associated invariant chain (Ii) interacts specifically with the ribosome-associated membrane protein 4 (RAMP4). RAMP4 is a small, tail-anchored protein of 66 amino acid residues that is homologous to the yeast YSY6 protein. YSY6 suppresses a secretion defect of a secY mutant in Escherichia coli. The interaction of RAMP4 with Ii occurred when nascent Ii chains reached a length of 170 amino acid residues and persisted until Ii chain completion, suggesting translocational pausing. Site-directed mutagenesis revealed that the region of Ii interacting with RAMP4 contains essential hydrophobic amino acid residues. Exchange of these residues for serines led to a reduced interaction with RAMP4 and inefficient N-glycosylation. We propose that RAMP4 controls modification of Ii and possibly also of other secretory and membrane proteins containing specific RAMP4-interacting sequences. Efficient or variable glycosylation of Ii may contribute to its capacity to modulate antigen presentation by MHC class II molecules.

Background: Selective protein import into the cell nucleus occurs in two steps: binding to the nuclear envelope, followed by energy-dependent transit through the nuclear pore complex. A 60 kD protein, importin, is essential for the first nuclear import step, and the small G protein Ran/TC4 is essential for the second. We have previously purified the 60 kD importin protein (importin 60) as a single polypeptide. Results We have identified importin 90, a 90 kD second subunit that dissociates from importin 60 during affinity chromatography on nickel (II)–nitrolotriacetic acid–Sepharose, a technique that was originally used to purify importin 60. Partial amino-acid sequencing of Xenopus importin 90 allowed us to clone and sequence its human homologue; the amino-acid sequence of importin 90 is strikingly conserved between the two species. We have also identified a homologous budding yeast sequence from a database entry. Importin 90 potentiates the effects of importin 60 on nuclear protein import, indicating that the importin complex is the physiological unit responsible for import. To assess whether nuclear localization sequences are recognized by cytosolic receptor proteins, a biotin-tagged conjugate of nuclear localization signals linked to bovine serum albumin was allowed to form complexes with cytosolic proteins in Xenopus egg extracts; the complexes were then retrieved with streptavidin–agarose. The pattern of bound proteins was surprisingly simple and showed only two predominant bands: those of the importin complex. We also expressed the human homologue of importin 60, Rch1p, and found that it was able to replace its Xenopus counterpart in a functional assay. We discuss the relationship of importin 60 and importin 90 to other nuclear import factors. Conclusion Importin consists of a 60 and a 90 kD subunit. Together, they constitute a cytosolic receptor for nuclear localization signals that enables import substrates to bind to the nuclear envelope.

We have purified a cytosolic protein from Xenopus eggs that is essential for selective protein import into the cell nucleus. The purified protein, named importin, promotes signal-dependent binding of karyophilic proteins to the nuclear envelope. We have cloned, sequenced, and expressed a corresponding cDNA. Importin shows 44% sequence identity with SRP1p, a protein associated with the yeast nuclear pore complex. Complete, signal-dependent import into HeLa nuclei can be reconstituted by combining importin purified from Xenopus eggs or expressed in E. coli with Ran/TC4. Evidence for additional stimulatory factors is provided.

Outgrowth of axons in the central nervous system is governed by specific molecular cues. Molecules detected so far act as ligands that bind to specific receptors. Here, we report a new membrane-associated lipid phosphate phosphatase that we have named plasticity-related gene 1 (PRG-1), which facilitates axonal outgrowth during development and regenerative sprouting. PRG-1 is specifically expressed in neurons and is located in the membranes of outgrowing axons. There, it acts as an ecto-enzyme and attenuates phospholipid-induced axon collapse in neurons and facilitates outgrowth in the hippocampus. Thus, we propose a novel mechanism by which axons are able to control phospholipid-mediated signaling and overcome the growth-inhibiting, phospholipid-rich environment of the extracellular space.

We study here the binding of ribosomes to the endoplasmic reticulum (ER) membrane and its dependence on nascent polypeptide-associated complex (NAC). For this, we use an in vitro translation system in combination with isolated microsomes. Importantly, all components in the system are derived from a single source, Saccharomyces cerevisiae. Ribosome nascent chains (RNCs) of the two naturally occurring invertase species (secreted or cytosolic) were prepared in wild-type, delta alpha NAC or delta alpha beta 1 beta 3 NAC translation lysates and tested for binding to the corresponding microsomal membranes. We provide evidence that NAC prevents binding of RNCs without a signal sequence to yeast membranes. In the absence of NAC, signal-less RNCs are able to bind to ER membranes. However, following puromycin treatment, only very few nascent chains translocate into the lumen, as detected by glycosylation.

The alpha-subunit of the TRAP complex (TRAP alpha) is a single-spanning membrane protein of the endoplasmic reticulum (ER) which is found in proximity of nascent polypeptide chains translocating across the membrane. Here, we demonstrate the widespread occurrence of TRAP alpha in eukaryotes as indicated by its existence in man, fish and plants. Despite the fact that the sequence homology is much lower than for other proteins in the translocation site, the overall topology, the location of the glycosylation sites and, most interestingly, the distribution of charges are conserved. These data indicate that the TRAP complex has a ubiquitous function.

Many proteins are transported through the ER membrane as they are synthesized. These include secretory proteins and proteins of the plasma membrane, of lysosomes, and of all organelles of the secretory pathway. Synthesis of these proteins begins in the cytoplasm, but they are then targeted to the ER membrane by signal sequences, which are characterized by a stretch of at least six apolar amino acids, and which are often located at the amino terminus of precursor molecules. Recognition of the signal sequence and targeting of the nascent chain generally requires the function of the signal recognition particle (SRP) and of its membrane receptor (also called docking protein), but alternative targeting pathways exist. The targeting phase is followed by the actual translocation process. Proposed mechanisms of translocation have ranged from the idea that the transport of a polypeptide chain occurs directly through the phopholipid bilayer without participation of membrane proteins, to models according to which polypeptides are transported through a hydrophilic or amphiphilic channel formed from transmembrane proteins (for discussion, see Rapoport 1991). It now appears that a protein-conducting channel does exist. The evidence comes from electrophysiological data and from the identification of membrane proteins as putative channel constituents. The present paper summarizes our knowledge on the components involved in the actual translocation process. The mechanisms by which polypeptides are targeted to the ER membrane have been discussed elsewhere (Rapoport 1992).

A chemically charged amber suppressor tRNA was used to introduce the photoactivatable amino acid (Tmd)Phe at a selected position within the signal sequence of the secretory protein preprolactin. This allowed the interactions of the NH2-terminal, the central, and the COOH-terminal regions of the signal sequence to be investigated during insertion into the membrane of the endoplasmic reticulum (ER). We found that different regions of the nascent chains were photocross-linked to different ER proteins. The TRAM protein (translocating chain-associating membrane protein) contacts the NH2-terminal region of the signal sequence while the mammalian Sec61p contacts the hydrophobic core of the signal sequence and regions COOH-terminal of this. These results suggest that the ER translocation complex is composed of heterologous protein subunits which contact distinct regions of nascent polypeptides during their membrane insertion.

To identify components of the mammalian endoplasmic reticulum involved in the translocation of secretory proteins, crosslinking and reconstitution methods were combined. A multispanning abundant membrane glycoprotein was found which is in proximity to nascent chains early in translocation. In reconstituted proteoliposomes, this protein is stimulatory or required for the translocation of secretory proteins.

Islets of Langerhans from carp were incubated with [U- 14 C]leucine at 15 °C for 2 h. About 3–4% of the newly synthesized protein was proinsulin. The identity of the newly synthesized protein was proved by: (1) its high specific radioactivity, (2) a molecular weight of 10 000, (3) co-chromatography with porcine insulin after tryptic digestion, (4) cleavage of the tryptic digest into two chains which co-chromatograph with the A- and B-chains of porcine insulin and (5) by the failure of cleavage of the original, non-trypsinated protein into two chains. A conversion of proinsulin into insulin was not observed.

(1) A simple procedure is described to separate reticulocytes of different maturity in high yield. (2) It is shown that exhaustion of supply of mitochondria susceptible to degradation by the lipoxygenase-ATP-dependent proteolysis system limits the extent of breakdown of mitochondria during in vitro maturation. The susceptibility of mitochondria depends on the maturity of the reticulocytes. (3) Incubation in the presence of calcium ions and calcium ionophore leads to full susceptibility of mitochondria in immature reticulocytes but has no effect on those in mature reticulocytes which are already fully susceptible to degradation. (4) Conditions which lead to rapid degradation of mitochondria do not affect the behaviour of the reticulocyte count. There appears to be no obligatory connection between the breakdown of mitochondria and of ribosomes.

The amino acid sequence of insulin of carp (Cyprinus carpio) has been determined and correlated with its biological activity in a fat-cell test and its structural properties as measured by circular dichroism and sedimentation analysis. The amino acid sequence of carp insulin displays some unusual features: the B chain is longer at the N ter- minus by two residues as compared with mammalian insulins and there are substitutions of the charged residues, found in most insulins at positions B21 and B22, by proline and threonine respectively. On the other hand, all amino acid residues essential for biological activity and for the association of insulin monomers are the same in carp insulin. Accordingly, the half-maximal response in a fat-cell test is reached with carp insulin at concentrations which are only three times higher than with porcine insulin and the maximal response is the same. The circular dichroism spectrum of carp insulin resembles greatly that of bovine insulin indicating that it has a similar spatial structure. Despite amino acid substitutions in the dimer-dimer contact region, carp insulin is able to form hexamers.

Publisher Summary This chapter discusses the biosynthesis of proinsulin of carp (cyprinus carpio). Islets of Langerhans of fish—Brockmann bodies—are suitable objects for the study of the biosynthesis of insulin. The islets of fish are much bigger than those of mammals and permit the isolation of relatively large amounts of mRNA. Therefore, it is possible to study the biosynthesis of insulin both on the cellular and subcellular level. The chapter discusses the primary sequence of carp insulin and reviews the possible structure-function relationships. Studies on the regulation of the biosynthesis of proinsulin and insulin in carp islets show that glucose has no effect whereas the temperature plays an essential role. Carp insulin may also serve as a model for the study of the biosynthesis of secretory proteins in general. Recently, the amino acid sequence of the carp insulin B-chain has been confirmed by sequencing on the nucleotide level.

The influence of temperature on the enzymatic breakdown of rRNA and ribosomal particles from rabbit reticulocytes by ribosomal ribonuclease and phosphatase was studied. Nucleosides were formed after the initial attack of ribonuclease on single-stranded regions of the RNA. The following conclusions were reached for free RNA: U and C were distributed in a random manner in the single-stranded loops at 20 °C. Short (A-U)-rich sequences were liberated at 37 °C, the relative share of which appeared to be higher in the 28-S RNA. It contained correspondingly longer G-C sequences. Clusters of A-U sequences of greater length than at 37 °C were melted at 50 °C. It was proved that only -U-U-U- sequences were split, by analysis of of the 3′ and 5′ terminal nucleotides of the primary split products of ribonuclease. The behaviour of the particle-bound RNA was modified considerably by interactions with proteins. Some of the cytosines appear to be protected against enzymatic attack at 20 °C. The 60-S subunit shows a strongly predominant melting of short U-A sequences at 37 °C. The reduction of the effective length of the U-A clusters may be due to interaction of the RNA with proteins. The 40-S subunit on the other hand would appear to exhibit a reduction of the effective length of G-C sequences. The interaction of the subunits with each other in an 80-S ribosome does not modify the behaviour of the subunits.