RAB2A: a major subacrosomal protein of bovine spermatozoa implicated in acrosomal biogenesis

The perinuclear theca (PT) of mammalian sperm is a unique subcellular structure encapsulating the nucleus. Compositionally, the PT is made up of at least six prominent polypeptides (60, 36, 31, 28, 24, and 15 kDa), of which only two have been sequence identified, as well as many less prominent ones. As an ongoing process in unveiling the protein composition of the PT, we have uncovered the sequence identity of the prominent 24-kDa polypeptide (PT24). Initial N-terminal sequence analysis obtained by Edman degradation suggested that PT24 is a RAB2 protein. This was corroborated by mass spectrometric analyses of trypsin-digested fragments of PT24, identifying RAB2A of the RAB2 subfamily as the best sequence match. Quadrapole/time-of-flight analysis identified 72%% sequence coverage between PT24 and bull, human, mouse, or rabbit RAB2A. Since a genome search only identified two RAB2 subfamily members, RAB2A and RAB2B, the 72%% sequence coverage of PT24 provides assurance that this protein is RAB2A and not a new RAB2 subfamily member. Furthermore, commercial RAB2A antibodies, raised against oligopeptide fragments in the unique C-terminal region of RAB2A, specifically labeled PT24 on Western blot analysis of PT extracts. These anti-RAB2A antibodies, along with immune serum that we raised and affinity purified against isolated PT24, demonstrated at both light and electron microscope levels that RAB2 is associated with the periphery of the growing proacrosomic and acrosomic vesicles in the Golgi and cap phases of spermiogenesis and consequently assembled as part of the PT. This pattern of subacrosomal assembly is reminiscent of the pathway used by SubH2Bv (PT15), another prominent and exclusive subacrosomal protein, indicating a common route for subacrosomal-PT assembly. Traditionally somatic RAB2 proteins are involved in vesicular transport between the endoplasmic reticulum and the cis-side of the Golgi apparatus. Our study suggests an unprecedented direction of RAB2A-mediated vesicular transport in spermatids during acrosomal biogenesis, from the trans-side of the Golgi apparatus to the nuclear envelope.     

A discrete repeated sequence defines a tubulin binding domain on microtubule-associated protein tau

The protein domain responsible for the interaction of tau with tubulin has been identified. Biophysical studies indicated that the synthetic peptide Val187-Gly204 (VRSKIG-STENLKHQPGGG) from the repetitive sequence on tau binds to two sites on the tubulin heterodimer and to one site on each of the microtubule-associated protein-interacting C-terminal tubulin peptides alpha(430-441) and beta(422-434). The binding data showed a relatively stronger interaction of Val187-Gly204 with beta(422-434) as compared to that with alpha(430-441). The interaction of this tau peptide with either alpha or beta tubulin peptides appears to be associated with conformational changes in both the tau and the tubulin peptides. The beta tubulin peptide also appears to induce a structural change of tau fragment Val218-Gly235. Interestingly, tau peptides Val187-Gly204 and Val218-Gly235 induced tubulin self-assembly in a cold-reversible fashion, and incorporated into the assembled polymers. The specificity of the interaction of the tau peptide was supported by the competition of tau protein for the interaction with the tubulin polymer. In addition, the tau peptide appears to contain the principal antigenic determinant(s) recognized by anti-idiotypic antibodies that react with the tubulin binding domains on microtubule-associated proteins. The present findings together with the demonstration of the presence of multiple sites for the binding of the alpha(430-441) and beta(422-434) tubulin fragments to tau, and the existence of repetitive sequences on tau, strongly support the hypothesis that the region of tau defined by the repetitive sequences is involved in its interaction with tubulin.     

Arabidopsis SAMT1 defines a plastid transporter regulating plastid biogenesis and plant development

S-Adenosylmethionine (SAM) is formed exclusively in the cytosol but plays a major role in plastids; SAM can either act as a methyl donor for the biogenesis of small molecules such as prenyllipids and macromolecules or as a regulator of the synthesis of aspartate-derived amino acids. Because the biosynthesis of SAM is restricted to the cytosol, plastids require a SAM importer. However, this transporter has not yet been identified. Here, we report the molecular and functional characterization of an Arabidopsis thaliana gene designated SAM TRANSPORTER1 (SAMT1), which encodes a plastid metabolite transporter required for the import of SAM from the cytosol. Recombinant SAMT1 produced in yeast cells, when reconstituted into liposomes, mediated the counter-exchange of SAM with SAM and with S-adenosylhomocysteine, the by-product and inhibitor of transmethylation reactions using SAM. Insertional mutation in SAMT1 and virus-induced gene silencing of SAMT1 in Nicotiana benthamiana caused severe growth retardation in mutant plants. Impaired function of SAMT1 led to decreased accumulation of prenyllipids and mainly affected the chlorophyll pathway. Biochemical analysis suggests that the latter effect represents one prominent example of the multiple events triggered by undermethylation, when there is decreased SAM flux into plastids.     

Interallelic complementation at the Drosophila melanogaster gastrulation defective locus defines discrete functional domains of the protein

The gastrulation defective (gd) locus encodes a novel serine protease that is involved in specifying the dorsal-ventral axis during embryonic development. Mutant alleles of gd have been classified into three complementation groups, two of which exhibit strong interallelic (intragenic) complementation. To understand the molecular basis of this interallelic complementation, we examined the complementation behavior of additional mutant alleles and sequenced alleles in all complementation groups. The data suggest that there are two discrete functional domains of Gd. A two-domain model of Gd suggesting that it is structurally similar to mammalian complement factors C2 and B has been previously proposed. To test this model we performed SP6 RNA microinjection to assay for activities associated with various domains of Gd. The microinjection data are consistent with the complement factor C2/B-like model. Site-directed mutagenesis suggests that Gd functions as a serine protease. An allele-specific interaction between an autoactivating form of Snake (Snk) and a gd allele altered in the protease domain suggests that Gd directly activates Snk in a protease activation cascade. We propose a model in which Gd is expressed during late oogenesis and bound within the perivitelline space but only becomes catalytically active during embryogenesis.     

Six2 defines and regulates a multipotent self-renewing nephron progenitor population throughout mammalian kidney development

Nephrons, the basic functional units of the kidney, are generated repetitively during kidney organogenesis from a mesenchymal progenitor population. Which cells within this pool give rise to nephrons and how multiple nephron lineages form during this protracted developmental process are unclear. We demonstrate that the Six2-expressing cap mesenchyme represents a multipotent nephron progenitor population. Six2-expressing cells give rise to all cell types of the main body of the nephron during all stages of nephrogenesis. Pulse labeling of Six2-expressing nephron progenitors at the onset of kidney development suggests that the Six2-expressing population is maintained by self-renewal. Clonal analysis indicates that at least some Six2-expressing cells are multipotent, contributing to multiple domains of the nephron. Furthermore, Six2 functions cell autonomously to maintain a progenitor cell status, as cap mesenchyme cells lacking Six2 activity contribute to ectopic nephron tubules, a mechanism dependent on a Wnt9b inductive signal. Taken together, our observations suggest that Six2 activity cell-autonomously regulates a multipotent nephron progenitor population.     

SAMP32, a testis-specific, isoantigenic sperm acrosomal membrane-associated protein

To identify novel human sperm membrane antigens, we analyzed two-dimensional gels of sperm extracts containing hydrophobic proteins that partitioned into Triton X-114. Four protein spots with isoelectric points (pIs) ranging from 4.5 to 5.5 and apparent molecular weights from 32 to 34 kDa were sequenced by mass spectrometry and found to contain common peptide sequences. Cloning the corresponding cDNA revealed that these protein spots were products of a single gene (SAMP32), encoding a protein of 32 kDa with a predicted pI of 4.57. SAMP32 has a potential transmembrane domain in the carboxyl terminus and is phosphorylated in vivo on serine 256. Northern blotting of eight human tissues and RNA dot blotting of 76 human tissues showed that SAMP32 expression was testis specific. SAMP32 contained an amino terminal domain homologous to the major malarial circumsporozoite surface protein and a domain similar to that of Krp1 from Schizosaccharomyces pombe in its carboxyl terminus. The SAMP32 locus consists of seven exons on chromosome 6q15-16.2. Antiserum against recombinant SAMP32 recognized protein spots originally cored from a two-dimensional gel. This antiserum strongly stained the equatorial segment and faintly stained the acrosome cap of ejaculated human spermatozoa by immunofluorescence. Immunoelectron microscopy showed that SAMP32 was associated with the inner acrosomal membrane in the principal and the equatorial segments of the sperm acrosome. By immunostaining enzyme-dissociated testicular cells, SAMP32 was localized to Golgi phase round spermatids and subsequent stages of acrosome biogenesis. Recombinant SAMP32 reacted with serum from an infertile man, suggesting that it is isoantigenic. Antibodies against recombinant SAMP32 inhibited both the binding and the fusion of human sperm to zona-free hamster eggs.     

Mouse sperm protein sp56 is a component of the acrosomal matrix

Previously, we identified the guinea pig sperm acrosomal matrix glycoprotein AM67 and demonstrated that it is most closely related to mouse sperm sp56, initially reported to be a cell-surface protein. On the contrary, our studies demonstrated that sp56 is an intra-acrosomal component. Based upon the homology between guinea pig AM67 and mouse sp56, we hypothesized that sp56 was part of the acrosomal matrix, a structure that had yet to be demonstrated to exist in mouse sperm. In this paper, we show that sp56 first appeared in late meiotic cells and accumulated during spermiogenesis, the haploid stage of spermatogenic cell development. Using affinity-purified anti-peptide antisera, we determined that the molecular weight of sp56 in cauda epididymal sperm approximated that of guinea pig AM67 ( approximately 67 000 M:(r)) and that sp56 was present in a high molecular weight, disulfide-linked complex. The forms of sp56 in pachytene spermatocytes and spermatids had higher molecular weights than was found for the sperm form; the size differences were apparently due to alterations in carbohydrate side chains. The sp56 complex could not be solubilized by the nonionic detergent Triton X-100 but remained associated with the dorsal surface of the mouse sperm head, demonstrating that sp56 is a component of the mouse sperm acrosomal matrix.     

Pulling geometry defines the mechanical resistance of a beta-sheet protein

Proteins show diverse responses when placed under mechanical stress. The molecular origins of their differing mechanical resistance are still unclear, although the orientation of secondary structural elements relative to the applied force vector is thought to have an important function. Here, by using a method of protein immobilization that allows force to be applied to the same all-beta protein, E2lip3, in two different directions, we show that the energy landscape for mechanical unfolding is markedly anisotropic. These results, in combination with molecular dynamics (MD) simulations, reveal that the unfolding pathway depends on the pulling geometry and is associated with unfolding forces that differ by an order of magnitude. Thus, the mechanical resistance of a protein is not dictated solely by amino acid sequence, topology or unfolding rate constant, but depends critically on the direction of the applied extension.     

Carbohydrate analysis throughout the development of a protein therapeutic

This review discusses the challenges involved in the characterization of the glycosylation of therapeutic glycoproteins. The focus is on methods that are most commonly used in regulatory filings and lot release testing of therapeutic glycoproteins. The different types of assays for carbohydrate analysis are reviewed, including the distinction between assays appropriate for lot release or better suited to testing during early drug development or in-depth characterization of the glycosylation. Characteristics of the glycoprotein and production process that should be considered when determining the amount of testing, the number of different methods to employ and when the testing should be performed during development of protein therapeutics is also discussed.     

Covariation of backbone motion throughout a small protein domain

The synchronization (correlation) of conformational fluctuations in folded proteins may influence the rates of enzyme catalysis and ligand binding as well as the stabilities of native proteins and their complexes. However, experimental detection of correlated motions remains difficult. Herein, we present an analysis of the covariation of NMR-derived backbone dynamical parameters among a family of ten mutants of a small protein. Both the spatial restriction and the time scales of backbone motions exhibit a higher degree of covariation than would be expected if the internal motions of each group were independent, providing experimental support for correlated dynamics. Application of this approach to other proteins may reveal dynamical correlations that influence catalysis, ligand-binding and/or protein stability.     

The Oxa2 protein of Neurospora crassa plays a critical role in the biogenesis of cytochrome oxidase and defines a ubiquitous subbranch of the Oxa1/YidC/Alb3 protein family

Proteins of the Oxa1/YidC/Alb3 family mediate the insertion of proteins into membranes of mitochondria, bacteria, and chloroplasts. Here we report the identification of a second gene of the Oxa1/YidC/Alb3 family in the genome of Neurospora crassa, which we have named oxa2. Its gene product, Oxa2, is located in the inner membrane of mitochondria. Deletion of the oxa2 gene caused a specific defect in the biogenesis of cytochrome oxidase and resulted in induction of the alternative oxidase (AOD), which bypasses the need for complex IV of the respiratory chain. The Oxa2 protein of N. crassa complements Cox18-deficient yeast mutants suggesting a common function for both proteins. The oxa2 sequence allowed the identification of a new subfamily of Oxa1/YidC/Alb3 proteins whose members appear to be ubiquitously present in mitochondria of fungi, plants, and animals including humans.     

PapD, a periplasmic transport protein in P-pilus biogenesis.

The product of the papD gene of uropathogenic Escherichia coli is required for the biogenesis of digalactoside-binding P pili. Mutations within papD result in complete degradation of the major pilus subunit, PapA, and of the pilinlike proteins PapE and PapF and also cause partial breakdown of the PapG adhesin. The papD gene was sequenced, and the gene product was purified from the periplasm. The deduced amino acid sequence and the N-terminal sequence obtained from the purified protein revealed that PapD is a basic and hydrophilic peripheral protein. A periplasmic complex between PapD and PapE was purified from cells that overproduced and accumulated these proteins in the periplasm. Antibodies raised against this complex reacted with purified wild-type P pili but not with pili purified from a papE mutant. In contrast, anti-PapD serum did not react with purified pili or with the culture fluid of piliated cells. However, this serum was able to specifically precipitate the PapE protein from periplasmic extracts, confirming that PapD and PapE were associated as a complex. It is suggested that PapD functions in P-pilus biogenesis as a periplasmic transport protein. Probably PapD forms complexes with pilus subunits at the outer surface of the inner membrane and transports them in a stable configuration across the periplasmic space before delivering them to the site(s) of pilus polymerization.     

Mapping the distribution of conformational information throughout a protein sequence

The three-dimensional structure of protein is encoded in the sequence, but many amino acid residues carry no essential conformational information, and the identity of those that are structure-determining is elusive. By circular permutation and terminal deletion, we produced and purified 25 Bacillus licheniformis beta-lactamase (ESBL) variants that lack 5-21 contiguous residues each, and collectively have 82% of the sequence and 92% of the non-local atom-atom contacts eliminated. Circular dichroism and size-exclusion chromatography showed that most of the variants form conformationally heterogeneous mixtures, but by measuring catalytic constants, we found that all populate, to a greater or lesser extent, conformations with the essential features of the native fold. This suggests that no segment of the ESBL sequence is essential to the structure as a whole, which is congruent with the notion that local information and modular organization can impart most of the tertiary fold specificity and cooperativity.     

MauG,a novel diheme protein required for tryptophan tryptophylquinone biogenesis

The biosynthesis of methylamine dehydrogenase (MADH) from Paracoccus denitrificans requires four genes in addition to those that encode the two structural protein subunits. None of these gene products have been previously isolated. One of these, mauG, exhibits sequence similarity to diheme cytochrome c peroxidases and is required for the synthesis of the tryptophan tryptophylquinone (TTQ) prosthetic group of MADH. A system was developed for the homologous expression of MauG in P. denitrificans. Its signal sequence was correctly processed, and it was purified from the periplasmic cell fraction. The protein contains two covalent c-type hemes, as predicted from the deduced sequence. EPR spectroscopy reveals that the protein as isolated possesses about equal amounts of one high-spin heme with axial symmetry and one low-spin heme with rhombic symmetry. The low-spin heme contains a major and minor component suggesting a small degree of heme heterogeneity. The high-spin heme and the major low-spin heme component each exhibit resonances that are atypical of c-type hemes and dissimilar to those reported for diheme cytochrome c peroxidases. MauG exhibited only very weak peroxidase activity when assayed with either c-type cytochromes or o-dianisidine as an electron donor. Fully reduced MauG was shown to bind carbon monoxide and could be reoxidized by oxygen. The relevance of these unusual properties of MauG is discussed in the context of its role in TTQ biogenesis.     

Vibrio cholerae FabV defines a new class of enoyl-acyl carrier protein reductase

Enoyl-acyl carrier protein (ACP) reductase catalyzes the last step of the fatty acid elongation cycle. The paradigm enoyl-ACP reductase is the FabI protein of Escherichia coli that is the target of the antibacterial compound, triclosan. However, some Gram-positive bacteria are naturally resistant to triclosan due to the presence of the triclosan-resistant enoyl-ACP reductase isoforms, FabK and FabL. The genome of the Gram-negative bacterium, Vibrio cholerae lacks a gene encoding a homologue of any of the three known enoyl-ACP reductase isozymes suggesting that this organism encodes a novel fourth enoyl-ACP reductase isoform. We report that this is the case. The gene encoding the new isoform, called FabV, was isolated by complementation of a conditionally lethal E. coli fabI mutant strain and was shown to restore fatty acid synthesis to the mutant strain both in vivo and in vitro. Like FabI and FabL, FabV is a member of the short chain dehydrogenase reductase superfamily, although it is considerably larger (402 residues) than either FabI (262 residues) or FabL (250 residues). The FabV, FabI and FabL sequences can be aligned, but only poorly. Alignment requires many gaps and yields only 15% identical residues. Thus, FabV defines a new class of enoyl-ACP reductase. The native FabV protein has been purified to homogeneity and is active with both crotonyl-ACP and the model substrate, crotonyl-CoA. In contrast to FabI and FabL, FabV shows a very strong preference for NADH over NADPH. Expression of FabV in E. coli results in markedly increased resistance to triclosan and the purified enzyme is much more resistant to triclosan than is E. coli FabI.     

Real-time observation of acrosomal dispersal from mouse sperm using GFP as a marker protein.

We produced transgenic mouse lines that accumulate mutated green fluorescent protein (EGFP) in sperm acrosome, a membrane limited organelle overlying the nucleus. The sperm showed normal fertilizing ability and the integrity of their acrosome was easily examined in a non-invasive manner by tracing the GFP in individual 'live' sperm with fluorescent microscopy. The time required for the dispersal of acrosomal contents was demonstrated to be approximately 3 s after the onset of acrosome reaction.