A membrane protein,TMCO5A,has a close relationship with manchette microtubules in rat spermatids during spermiogenesis

We isolated the transmembrane and coiled‐coil domains 5A (Tmco5A) gene using polymerase chain reaction‐based subtraction technique and showed that Tmco5A was predominantly expressed in rat testes starting at 4 weeks of postnatal development. When expressed in COS7 cells, TMCO5A was found to be distributed in the endoplasmic reticulum‐nuclear membrane (ER‐NM) of cells as a membrane‐associated protein, while TMCO5AΔC lacking the transmembrane region (TM) mislocalized and diffused throughout the cytoplasm. The result suggested that TM is responsible for the retention of TMCO5A at the ER‐NM. Immunocytochemical and immunoblotting analyses indicated that TMCO5A was localized along the posterior part of the nuclei in both round and elongated rat spermatids but disappeared from epididymal spermatozoa. Double immunolabeling of isolated spermatids with the anti‐TMCO5A and the anti‐β tubulin antibodies showed that TMCO5A was always found to be closely associated with developing manchette microtubules but did not completely colocalize with them. On the other hand, we found that almost all TMCO5A colocalized with SUN4, a linker of nucleoskeleton and cytoskeleton complex protein present at the posterior part of spermatid nuclei. These data suggested that TMCO5A is located closer to the nuclei than the manchette microtubules. It is likely that TMCO5A, in association with manchette microtubules, is involved in the process of spermiogenesis.     

Ran,a GTP-binding protein involved in nucleocytoplasmic transport and microtubule nucleation,relocates from the manchette to the centrosome region during rat spermiogenesis

Ran, a Ras-related GTPase, is required for transporting proteins in and out of the nucleus during interphase and for regulating the assembly of microtubules. cDNA cloning shows that rat testis, like mouse testis, expresses both somatic and testis-specific forms of Ran-GTPase. The presence of a homologous testis-specific form of Ran-GTPase in rodents implies that the Ran-GTPase pathway plays a significant role during sperm development. This suggestions is supported by distinct Ran-GTPase immunolocalization sites identified in developing spermatids. Confocal microscopy demonstrates that Ran-GTPase localizes in the nucleus of round spermatids and along the microtubules of the manchette in elongating spermatids. When the manchette disassembles, Ran-GTPase immunoreactivity is visualized in the centrosome region of maturing spermatids. The circumstantial observation that fractionated manchettes, containing copurified centrin-immunoreactive centrosomes, can organize a three-dimensional lattice in the presence of taxol and GTP, points to the role of Ran-GTPase and associated factors in microtubule nucleation as well as the potential nucleating function of spermatid centrosomes undergoing a reduction process. Electron microscopy demonstrates the presence in manchette preparations of spermatid centrosomes, recognized as such by their association with remnants of the implantation fossa, a dense plate observed only at the basal surface of developing spermatid and sperm nuclei. In addition, we have found importin beta1 immunoreactivity in the nucleus of elongating spermatids, a finding that, together with the presence of Ran-GTPase in the nucleus of round spermatids and the manchette, suggest a potential role of Ran-GTPase machinery in nucleocytoplasmic transport. Our expression and localization analysis, correlated with functional observations in other cell systems, suggest that Ran-GTPase may be involved in both nucleocytoplasmic transport and microtubules assembly, two critical events during the development of functional sperm. In addition, the manchette-to-centrosome Ran-GTPase relocation, together with the similar redistribution of various proteins associated to the manchette, suggest the existence of an intramanchette molecular transport mechanism, which may share molecular analogies with intraflagellar transport.     

Isolation and characterization of the spermatid-specific Smrp1 gene encoding a novel manchette protein

The manchette, which is the structure that appears around the nuclei of elongated spermatids, is assumed to be involved in nuclear shaping during spermiogenesis and the transport of various proteins between the nucleus and sperm tail. In this report, we describe the molecular cloning and characterization of a mouse spermatid-specific manchette-related protein 1 (Smrp1) from a spermatid-specific subtracted mouse testis cDNA library. The isolated Smrp1 cDNA clones could be divided into three variants based on sequence analysis. Computer-assisted analysis showed that these variants were splice variants from a single locus of the mouse genome. The three putative proteins consisted of 296, 260, and 175 amino acids, respectively. Although 155 amino acids of the N terminus were common to the three proteins, they were distinguished by their C-terminal regions. Western blot analyses using specific antisera showed that SMRP1 expression was specific to the testes and that only the 261-amino-acid form was translated into protein. Immunohistochemistry revealed that SMRP1 was localized to the cytoplasm of step 9-12 elongated spermatids. The protein appeared in a cap formation that covered the caudal sides of the elongated nuclei. This localization pattern coincided with that of the manchette. SMRP1 may play an important role as a functional protein that co-operates with manchette proteins.     

A protein encoded by a new family of mobile elements from Euryarchaea exhibits three domains with novel folds

We present here the 2.6Å resolution crystal structure of the pT26‐6p protein, which is encoded by an ORF of the plasmid pT26‐2, recently isolated from the hyperthermophilic archaeon, Thermococcus sp. 26,2. This large protein is present in all members of a new family of mobile elements that, beside pT26‐2 include several virus‐like elements integrated in the genomes of several Thermococcales and Methanococcales (phylum Euryarchaeota). Phylogenetic analysis suggested that this protein, together with its nearest neighbor (organized as an operon) have coevolved for a long time with the cellular hosts of the encoding mobile element. As the sequences of the N and C‐terminal regions suggested a possible membrane association, a deletion construct (739 amino acids) was used for structural analysis. The structure consists of two very similar β‐sheet domains with a new topology and a five helical bundle C‐terminal domain. Each of these domains corresponds to a unique fold that has presently not been found in cellular proteins. This result supports the idea that proteins encoded by plasmid and viruses that have no cellular homologues could be a reservoir of new folds for structural genomic studies.     

Mammalian Ddi2 is a shuttling factor containing a retroviral protease domain that influences binding of ubiquitylated proteins and proteasomal degradation

Although several proteasome subunits have been shown to bind ubiquitin (Ub) chains, many ubiquitylated substrates also associate with 26S proteasomes via “shuttling factors.” Unlike the well-studied yeast shuttling factors Rad23 and Dsk2, vertebrate homologs Ddi2 and Ddi1 lack a Ub-associated domain; therefore, it is unclear how they bind Ub. Here, we show that deletion of Ddi2 leads to the accumulation of Ub conjugates with K11/K48 branched chains. We found using affinity copurifications that Ddi2 binds Ub conjugates through its Ub-like domain, which is also required for Ddi2 binding to proteasomes. Furthermore, in cell extracts, adding Ub conjugates increased the amount of Ddi2 associated with proteasomes, and adding Ddi2 increased the binding of Ub conjugates to purified proteasomes. In addition, Ddi2 also contains a retroviral protease domain with undefined cellular roles. We show that blocking the endoprotease activity of Ddi2 either genetically or with the HIV protease inhibitor nelfinavir increased its binding to Ub conjugates but decreased its binding to proteasomes and reduced subsequent protein degradation by proteasomes leading to further accumulation of Ub conjugates. Finally, nelfinavir treatment required Ddi2 to induce the unfolded protein response. Thus, Ddi2 appears to function as a shuttling factor in endoplasmic reticulum–associated protein degradation and delivers K11/K48-ubiquitylated proteins to the proteasome. We conclude that the protease activity of Ddi2 influences this shuttling factor activity, promotes protein turnover, and helps prevent endoplasmic reticulum stress, which may explain nelfinavir’s ability to enhance cell killing by proteasome inhibitors.     

Evidence that selenium in rat sperm is associated with a cysteine-rich structural protein of the mitochondrial capsules

The keratinous capsules surrounding rat sperm mitochondria were isolated 24 days after intratesticular injections of [⁷⁵Se] selenite or [³⁵S] cysteine. Dodecyl sulfate-polyacrylamide gel electrophoresis of purified, doubly labeled mitochondrial capsules revealed only a single ⁷⁵Se-labeled component, whose molecular weight was 17,000, in agreement with previously reported observations obtained with cruder sperm fractions. Most of the ³⁵S label and the major zone of stained protein on the gels coincided with the position of ⁷⁵Se, suggesting that selenium is associated with a cysteine-rich structural protein. The level of selenium in rat sperm, 195 ± 3.2 ng/10⁸ sperm (approximately 30 ppm), determined by hydride generation and atomic absorption spectrophotometry, is consistent with a structural function for this trace element in the sperm.     

A non-synonymous mutation in a conserved site of the MTTP gene is strongly associated with protein activity and fatty acid profile in pigs

Despite the economic interest of the fatty acid profile in pigs, no gene has been convincingly associated with this trait so far. Here, the porcine microsomal triglyceride transfer protein ( MTTP ) gene, which plays a crucial role in the assembly of nascent lipoproteins, has been analysed as a positional candidate gene for a QTL affecting the fatty acid composition that was previously identified on chromosome 8 in an Iberian by Landrace F₂ cross. By resequencing a panel of different breeds, a non-synonymous polymorphism in a conserved residue of the lipid transfer domain of MTTP was identified. Association analyses with this polymorphism showed a strong association with the fatty acid composition of porcine fat, much stronger than the QTL effect, in the F₂ cross and in a synthetic Sino-European line. In addition, in vitro activity assays in liver protein extracts have shown that this mutation is also associated with the lipid transfer activity of the MTTP protein ( P  <   0.1). These results suggest that the detected polymorphism is a potential causal factor of the fatty acid composition QTL. There appears to be an interaction between the porcine MTTP genotype and the type of fat source in the pig diet, which would agree with the previous results on the biology of MTTP biology.     

The ATPase and helicase activities of Prp43p are stimulated by the G‐patch protein Pfa1p during yeast ribosome biogenesis

Prp43p is a RNA helicase required for pre‐mRNA splicing and for the synthesis of large and small ribosomal subunits. The molecular functions and modes of regulation of Prp43p during ribosome biogenesis remain unknown. We demonstrate that the G‐patch protein Pfa1p, a component of pre‐40S pre‐ribosomal particles, directly interacts with Prp43p. We also show that lack of Gno1p, another G‐patch protein associated with Prp43p, specifically reduces Pfa1p accumulation, whereas it increases the levels of the pre‐40S pre‐ribosomal particle component Ltv1p. Moreover, cells lacking Pfa1p and depleted for Ltv1p show strong 20S pre‐rRNA accumulation in the cytoplasm and reduced levels of 18S rRNA. Finally, we demonstrate that Pfa1p stimulates the ATPase and helicase activities of Prp43p. Truncated Pfa1p variants unable to fully stimulate the activity of Prp43p fail to complement the 20S pre‐rRNA processing defect of Δpfa1 cells depleted for Ltv1p. Our results strongly suggest that stimulation of ATPase/helicase activities of Prp43p by Pfa1p is required for efficient 20S pre‐rRNA‐to‐18S rRNA conversion.     

Antigenicity of the region encoded by exon8 of the human serine protease, HtrA2/Omi, is associated with its protein solubility

HtrA2/Omi, a mitochondrial serine protease, is pivotal in regulating apoptotic cell death. To determine the location of antigenic determinants in HtrA2/Omi, we expressed a series of the N-terminally truncated HtrA2/Omi as GST fusion proteins in E. coli. We assessed protein solubility and antigenic reactivity of various N-terminally truncated HtrA2/Omi proteins by binding to glutathione beads and immunoblot analyses, respectively. We identified that the region encoded by exon8 of HtrA2/Omi was expressed as a highly soluble form and contains an antigenic determinant specifically recognized by a polyclonal serum against HtrA2/Omi. Our data provide evidence that protein solubility of the specific region in target proteins may contribute to the antigenicity.     

Gemin3: A novel DEAD box protein that interacts with SMN, the spinal muscular atrophy gene product, and is a component of gems

The survival of motor neurons (SMN) gene is the disease gene of spinal muscular atrophy (SMA), a common motor neuron degenerative disease. The SMN protein is part of a complex containing several proteins, of which one, SIP1 (SMN interacting protein 1), has been characterized so far. The SMN complex is found in both the cytoplasm and in the nucleus, where it is concentrated in bodies called gems. In the cytoplasm, SMN and SIP1 interact with the Sm core proteins of spliceosomal small nuclear ribonucleoproteins (snRNPs), and they play a critical role in snRNP assembly. In the nucleus, SMN is required for pre-mRNA splicing, likely by serving in the regeneration of snRNPs. Here, we report the identification of another component of the SMN complex, a novel DEAD box putative RNA helicase, named Gemin3. Gemin3 interacts directly with SMN, as well as with SmB, SmD2, and SmD3. Immunolocalization studies using mAbs to Gemin3 show that it colocalizes with SMN in gems. Gemin3 binds SMN via its unique COOH-terminal domain, and SMN mutations found in some SMA patients strongly reduce this interaction. The presence of a DEAD box motif in Gemin3 suggests that it may provide the catalytic activity that plays a critical role in the function of the SMN complex on RNPs.     

A rubrerythrin-like oxidative stress protein of Clostridium acetobutylicum is encoded by a duplicated gene and identical to the heat shock protein Hsp21

Comparison of the N-terminus of the heat shock protein Hsp21 of Clostridium acetobutylicum with proteins predicted to be encoded by the genome of this bacterium revealed that this stress protein is encoded by two almost identical open reading frames CAC3597 and CAC3598. These genes encode a rubrerythrin-like protein with the rubredoxin-like FeS4 domain at the N-terminus and the ferritin-like diiron domain (rubrerythrin domain) at the C-terminus. Thus, the order of the two putative functional domains is reversed compared to "normal" rubrerythrins. This protein is proposed to be involved in the oxidative stress response of strict anaerobic bacteria. Northern blot analysis indicated that hsp21 is induced by heat and oxidative stress (air, H2O2). Hsp21 of C. acetobutylicum can be considered as a "reverse" rubrerythrin and a role of this stress protein, which is conserved among clostridia and other strict anaerobic bacteria, in the heat and oxidative stress response is proposed.     

Proteins with tandemly arranged repeats of a highly charged 16-amino-acid motif encoded by the Dhmst101 gene family are structural components of the outer sheath of the extremely elongated sperm tails of Drosophila hydei

Fruit fly species of the genus Drosophila show a remarkable variation in sperm length. Some of them produce gigantic sperm several times the total male body length. Sperm of Drosophila hydei, for example, are more than 20 mm long. Little is known about the advantage of such elongated sperm or about the proteins that stabilize their thin flagellar tails. Recently, two members of a novel gene family Dhmst101(1) and Dhmst101(2), whose gene products are associated with the sperm tail, were isolated and characterized. Here a third member of this gene family, Dhmst101(3), is described. It was previously demonstrated that all three genes are located in a single small cluster on chromosome 5 of D. hydei. They are located within 15 kb of genomic DNA, oriented in the same direction and transcribed testis-specifically. The encoded sperm tail-specific proteins are mainly composed of tandemly arranged repeats of a highly charged, cysteine-containing motif of 16 amino acids with the consensus sequence KKKCA/EEAAKKEKEAAE. Experiments with synthetic repeat monomers and dimers have demonstrated a tendency for alpha-helical rod formation, which increased strongly with an increase in repeat number. Therefore, Dhmst101 proteins with 7-60 repeats with regularly spaced cystein-residues are thus expected to form long alpha-helical rods cross-linked by numerous Cys-Cys bridges. Here we apply immunoelectron microscopy and monospecific antibodies, alpha-mst101, raised against the KKKCAEAAKKEKEAAE-motif to investigate the distribution of Dhmst101 proteins within the sperm tail of D. hydei. We show that Dhmst101 proteins are part of the outer sheath of the sperm tail where they presumably help to provide a tight but elastic envelope for the extremely extended spermatozoa of D. hydei.     

Expression pattern and raft association of NIPSNAP3 and NIPSNAP4, highly homologous proteins encoded by genes in close proximity to the ATP-binding cassette transporter A1

The highly homologous genes NIPSNAP3 and NIPSNAP4, with 87% amino acid identity, are members of the NIPSNAP family with putative roles in vesicular trafficking. NIPSNAP3 mRNA and NIPSNAP4 mRNA and protein were detected in multiple tissues and cells at varying degrees. Interestingly, NIPSNAP3 is most highly expressed in skeletal muscle, where NIPSNAP4 has a low mRNA abundance. NIPSNAP4 was found associated with membranes and partly localized in rafts. The ubiquitous expression of the highly conserved NIPSNAPs and their association with membranes further support an important cellular function of these proteins probably linked to vesicular trafficking. The NIPSNAP3 and NIPSNAP4 genes are located in close proximity to the 3' end of the ATP-binding cassette transporter A1 (ABCA1), whose mutations cause familial high-density lipoprotein deficiency syndromes. The adjacent genomic location and the finding that ABCA1 is a regulator of vesicular trafficking may indicate a functional relation of these proteins, even though NIPSNAP4 does not interact directly with ABCA1 nor is its expression altered in cells with mutated ABCA1.