Hybrid character of a large neurofilament protein (NF-M): intermediate filament type sequence followed by a long and acidic carboxy-terminal extension

The sequence of the amino-terminal 436 residues of porcine neurofilament component NF-M (apparent mol. wt. in gel electrophoresis 160 kd), one of the two high mol. wt. components of mammalian neurofilaments, reveals the typical structural organization of an intermediate filament (IF) protein of the non-epithelial type. A non-alpha-helical arginine-rich headpiece with multiple beta-turns (residues 1-98) precedes a highly alpha-helical rod domain able to form double-stranded coiled-coils (residues 99-412) and a non-alpha-helical tailpiece array starting at residue 413. All extra mass of NF-M forms, as a carboxy-terminal tailpiece extension of approximately 500 residues, an autonomous domain of unique composition. Limited sequence data in the amino-terminal region of this domain document a lysine- and particularly glutamic acid-rich array somewhat reminiscent of the much shorter tailpiece extension of NF-L (apparent mol. wt. 68 kd), the major neurofilament protein. NF-M is therefore a true intermediate filament protein co-polymerized with NF-L via presumptive coiled-coil type interactions and not a peripherally bound associated protein of a filament backbone built exclusively from NF-L. Along the structurally conserved coiled-coil domains the two neurofilament proteins show only approximately 65% sequence identity, a value similar to that seen when NF-L and NF-M are compared with mesenchymal vimentin. The highly charged and acidic tailpiece extensions of all triplet proteins particularly rich in glutamic acid seem unique to the neurofilament type of IFs. They could form extra-filamentous scaffolds suitable for interactions with other neuronal components.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of a cDNA for the rat heavy neurofilament polypeptide (NF-H)

We have isolated from a rat brain lambda gt11 expression library two overlapping cDNA clones of sizes 2.5 and 3.0 kb corresponding to the heavy neurofilament polypeptide (NF-H). The 2.5 kb insert apparently represents virtually the whole of the C-terminal tail, the 3.0 kb insert also encodes the conserved epitope for the monoclonal antibody, anti-IFA. The identity of the cDNAs was established by comparison of the predicted amino acid sequence with the known partial amino acid sequence of porcine NF-H. A repeat peptide sequence that may be a multiphosphorylation site was identified in the C-terminal non-helical tail.     

Identification and characterization of a unique Xenopus laevis egg envelope component,ZPD

We report the identification of a previously undetected Xenopus laevis egg envelope component discovered through cloning experiments. A cDNA sequence was found that represented a mature protein of 32 kDa. Peptide antibodies were generated to probe for the protein in egg envelope samples and reactivity was found to a glycoprotein of approximately 80 kDa. When deglycosylated egg envelope samples were probed, a 32 kDa protein was labeled, confirming the size of the translated cDNA sequence. A BLAST analysis showed that it is most closely related (34% amino acid identity) to the ZP domains of mammalian tectorin, uromodulin and ZPA. From a dendrogram of known egg envelope glycoproteins, the new glycoprotein was shown to be unique among egg envelope components and was designated ZPD. A similar glycoprotein was identified by immunocrossreactivity in Xenopus tropicalis and Xenopus borealis egg envelopes.     

Identification and characterization of a novel mammalian intermediate filament-associated protein

A novel monoclonal antibody, designated M1.4, recognizes the high molecular weight microtubule-associated protein MAP1A (ca. Mr 380 kD) in both bovine and rat brain. In HeLa cells, however, M1.4 binds to a 240 kD polypeptide on immunoblots and co-localizes with both vimentin and cytokeratin filaments using double-label immunofluorescence microscopy. Immunoelectron microscopy indicates that the 240 kD polypeptide localizes along bundled intermediate filaments in a periodic manner. Two-dimensional electrophoretic analysis indicates that the 240 kD polypeptide has a basic pI of 7.7. When HeLa cell intermediate filaments are isolated using standard non-ionic detergent/high-salt conditions the 240 kD polypeptide does not sediment with the intermediate filaments, unlike the established intermediate filament-associated protein plectin. Immunoblot analysis with M1.4 shows the 240 kD polypeptide is expressed in a number of mammalian cell lines. Additionally, double-label immunofluorescence shows the 240 kD polypeptide to associate with vimentin filaments in African Green Monkey kidney (CV-1) and JC neuroblastoma cells. Due to its unique biochemical and biological characteristics, the 240 kD polypeptide is clearly a novel intermediate filament-associated protein for which we have proposed the designation gyronemin (Gr. gyros: around; nemin: filament).     

Cleavage of human fibroblast type I procollagen by mammalian collagenase: demonstration of amino- and carboxy-terminal extension peptides.

Human skin fibroblasts in monolayer culture synthesize and secrete precursor forms of collagen into the culture medium. The type I collagen precursor, the major precursor in the culture medium, was isolated on DEAE cellulose chromatography and subjected to mammalian collagenase cleavage. The amino terminal cleavage fragments had a higher molecular weight than α1^A and α2^A, but did not contain interchain disulfide bonds. The carboxy-terminal cleavage fragments formed high molecular weight aggregates which contained interchain disulfide bonds. These results indicate that human type I procollagen contains noncollagenous amino and carboxy-terminal extension peptides and that all of the interchain disulfide bonds are on the carboxy-terminal portion of the molecule.     

Isolation and characterization of monkey interphotoreceptor retinoid-binding protein, a unique extracellular matrix component of the retina

The interphotoreceptor retinoid-binding protein (IRBP) has been isolated from monkey interphotoreceptor matrix (IPM). Following gentle washing of the IPM from the retinal surface, the protein was purified to homogeneity by concanavalin A-Sepharose affinity chromatography, ion-exchange high-performance liquid chromatography (HPLC), and size-exclusion HPLC. Bovine IRBP was purified similarly and compared with the monkey protein. Sedimentation equilibrium analysis yielded a molecular weight of 106 000 +/- 2900 for the native monkey protein. Sedimentation velocity analysis gave a sedimentation coefficient of 5.4 +/- 0.3 S and a frictional ratio of 1.59, indicating an asymmetrical molecular shape. IRBP contains neutral sugar, including fucose, and sialic acid; the glycoprotein nature of the proteins probably accounts for the microheterogeneity observed in the electrofocusing pattern of both bovine and monkey IRBP. Both IRBPs have isoelectric points between 6.0 and 7.0. The fluorescence emission lambda max of the bound ligand was 470 nm with excitation at 340 nm, while the excitation lambda max was 333 nm with emission at 470 nm, for monkey IRBP incubated with exogenous all-trans-retinol. The amino acid compositions of the monkey and bovine proteins are similar; nonpolar amino acids account for over 50% of the residues, which may explain the apparent hydrophobic nature of the isolated proteins. The amino-terminal analyses indicated considerable homology between the monkey and bovine IRBPs in this region and verified the purity of the isolated proteins. IRBP thus appears to be a unique, conserved glycoprotein of the retinal extracellular matrix that could serve as a retinoid-transport vehicle.     

Self-assembly in vitro of the 68,000 molecular weight component of the mammalian neurofilament triplet proteins into intermediate-sized filaments

The mammalian neurofilament triplet proteins (210, 160 and 68 × 10³M_r proteins) are resolved by anion exchange chromatography in the presence of urea. Upon dialysis against physiological buffers at 37 °C only the 68 × 10³M_r protein shows self-assembly into morphologically normal intermediate-sized filaments. Addition of 210 × 10³M_r protein to 68 × 10₃M_r protein leads to shorter filaments, which upon embedding reveal a rough surface and whisker-like protrusions that are not present on the smooth surface of filaments assembled from 68 × 10₃M_r protein alone. Certain emerging principles of neurofilament structure are discussed, emphasizing a possible relation between neurofilaments and other intermediate-sized filaments.     

hFis1, a novel component of the mammalian mitochondrial fission machinery

The balance between the fission and fusion mechanisms regulate the morphology of mitochondria. In this study we have identified a mammalian protein that we call hFis1, which is the orthologue of the yeast Fis1p known to participate in yeast mitochondrial division. hFis1, when overexpressed in various cell types, localized to the outer mitochondrial membrane and induced mitochondrial fission. This event was inhibited by a dominant negative mutant of Drp1 (Drp1(K38A)), a major component of the fission apparatus. Fragmentation of the mitochondrial network by hFis1 was followed by the release of cytochrome c and ultimately apoptosis. Bcl-xL was able to block cytochrome c release and apoptosis but failed to prevent mitochondrial fragmentation. Our studies show that hFis1 is part of the mammalian fission machinery and suggest that regulation of the fission processes might be involved in apoptotic mechanisms.     

Repetitious and unique sequences in the heterogeneous nuclear and cytoplasmic messenger RNA of mammalian and insect cells

Initiation of DNA synthesis has been followed in mouse myeloma cells grown in suspension culture. In cells labeled with ³H-thymidine for short times, label first appears in short fragments of DNA which can be chased into bulk DNA (>50 S) upon further incubation in unlabeled thymidine. In a 15 min pulse, DNA fragments with a sedimentation coefficient of 30 S tend to accumulate. Our results support the contention that DNA synthesis is discontinuous in myeloma cells.However, a search for RNA associated with nascent DNA in the myeloma system was unsuccessful. Newly synthesized DNA was isolated on a benzoylated naphthoylated DEAE cellulose column. After heat denaturation, this fraction was centrifuged to equilibrium in a Cs₂SO₄ density gradient. The nascent DNA displays no shift in density greater than the density of the bulk DNA. When cells were pulse labeled with ³H-uridine and the nascent DNA fraction analyzed on Cs₂SO₄ density gradients, no ³H-labeled RNA was found associated with the DNA peak or at intermediate densities that would be indicative of a RNA-DNA molecule, covalently linked. Unless scission of the RNA primers occurs immediately after the initiation of DNA synthesis, our results indicate that DNA synthesis commences without RNA primers in myeloma cells.     

Cloning and functional characterization of mammalian homologues of the COPII component Sec23.

We screened a human cDNA library with a probe derived from a partial SEC23 mouse homologue and isolated two different cDNA clones (hSec23A and hSec23B) encoding proteins of a predicted molecular mass of 85 kDa. hSec23Ap and hSec23Bp were 85% identical and shared 48% identity with the yeast Sec23p. Affinity-purified anti-hSec23A recognized a protein of approximately 85 kDa on immunoblots of human, mouse, and rat cell extracts but did not recognize yeast Sec23p. Cytosolic hSec23Ap migrated with an apparent molecular weight of 350 kDa on a gel filtration column, suggesting that it is part of a protein complex. By immunoelectron microscopy, hSec23Ap was found essentially in the ribosome-free transitional face of the endoplasmic reticulum (ER) and associated vesicles. hSec23Ap is a functional homologue of the yeast Sec23p as the hSec23A isoform complemented the temperature sensitivity of the Saccharomyces cerevisiae sec23-1 mutation at a restrictive temperature of 34 degrees C. RNase protection assays indicated that both hSec23 isoforms are coexpressed in various human tissues, although at a variable ratio. Our data demonstrate that hSec23Ap is the functional human counterpart of the yeast COPII component Sec23p and suggest that it plays a similar role in mammalian protein export from the ER. The exact function of hSec23Bp remains to be determined.     

Repair of UV-damaged DNA in mammalian skin followed by the immunohistochemical method

DNA repair in murine and guinea pig skin has been studied by the immunohistochemical method. For the detection of DNA photolesions in situ by the indirect immunofluorescence (IF) method two antisera directed against DNA-pyrimidine-dimers and DNA-psoralen-photoadducts have been applied. The IF assay enabled to detect the DNA photodamage induced by high UV-doses, exceeding more than fivefold minimal phototoxic response of the skin. It was found that IF staining gradually disappeared due to DNA repair, and at 48 h after UV-exposure both types of the DNA photolesions were no more detectable. Importantly, the IF method revealed that the persistence of DNA-pyrimidine-dimers could be traced for a longer time than that detectable by UV-endonuclease incision method.     

Novel rearrangements of herpes simplex virus DNA sequences resulting from duplication of a sequence within the unique region of the L component.

We constructed insertion mutants of herpes simplex virus type 1 that contained a duplication of DNA sequences from the BamHI-L fragment (map units 0.706 to 0.744), which is located in the unique region of the L component (UL) of the herpes simplex virus type 1 genome. The second copy of the BamHI-L sequence was inserted in inverted orientation into the viral thymidine kinase gene (map units 0.30 to 0.32), also located within UL. A significant fraction of the progeny produced by these insertion mutants had genomes with rearranged DNA sequences, presumably resulting from intramolecular or intermolecular recombination between the BamHI-L sequences at the two different genomic locations. The rearranged genomes either had an inversion of the DNA sequence flanked by the duplication or were recombinant molecules in which different regions of the genome had been duplicated and deleted. Genomic rearrangements similar to those described here have been reported previously but only for herpes simplex virus insertion mutants containing an extra copy of the repetitive a sequence. Such rearrangements have not been reported for insertion mutants that contain duplications of herpes simplex virus DNA sequences from largely unique regions of the genome. The implications of these results are discussed.     

RNF17, a component of the mammalian germ cell nuage, is essential for spermiogenesis

Nuages are found in the germ cells of diverse organisms. However, nuages in postnatal male germ cells of mice are poorly studied. Previously, we cloned a germ cell-specific gene named Rnf17, which encodes a protein containing both a RING finger and tudor domains. Here, we report that RNF17 is a component of a novel nuage in male germ cells--the RNF17 granule, which is an electron-dense non-membrane bound spherical organelle with a diameter of 0.5 mum. RNF17 granules are prominent in late pachytene and diplotene spermatocytes, and in elongating spermatids. RNF17 granules are distinguishable from other known nuages, such as chromatoid bodies. RNF17 is able to form dimers or polymers both in vitro and in vivo, indicating that it may play a role in the assembly of RNF17 granules. Rnf17-deficient male mice were sterile and exhibited a complete arrest in round spermatids, demonstrating that Rnf17 encodes a novel key regulator of spermiogenesis. Rnf17-null round spermatids advanced to step 4 but failed to produce sperm. These results have shown that RNF17 is a component of a novel germ cell nuage and is required for differentiation of male germ cells.     

Sept12 is a component of the mammalian sperm tail annulus

Since their essential role in cytokinesis was first shown in yeast, the septins have been described to function in diverse cellular contexts. The members of this unique class of GTPases are capable of binding and hydrolyzing GTP, associating with membranes and oligomerizing into higher order structures. Here we describe Sept12, a novel septin, identified in a yeast two hybrid screen using Sept5 as the bait. Sept12 contains the primary sequence elements of a septin and is capable of interacting with other septins. In addition, Sept12 purifies with bound nucleotide and binds to phosphoinositides, confirming its identity as a septin. RT-PCR and Northern blots reveal that Sept12 mRNA is expressed predominantly in testis, and this is supported by tissue Western blots. In rats, Sept12 protein levels rise upon sexual maturity and the Sept12 protein colocalizes with the annulus in isolated mature spermatozoa. Further, coexpression of Sept12 with Sept4, an essential annulus component, results in complete colocalization of both proteins into robust and highly curved filaments in CHO cells. This study suggests Sept12 may be involved in mammalian fertility.