Characterization of different oligomeric forms of CRISP2 in the perinuclear theca versus the fibrous tail structures of boar spermatozoa†.

Mammalian sperm carry a variety of highly condensed insoluble protein structures such as the perinuclear theca, the fibrous sheath and the outer dense fibers, which are essential to sperm function. We studied the role of cysteine rich secretory protein 2 (CRISP2); a known inducer of non-pathological protein amyloids, in pig sperm with a variety of techniques. CRISP2, which is synthesized during spermatogenesis, was localized by confocal immunofluorescent imaging in the tail and in the post-acrosomal region of the sperm head. High-resolution localization by immunogold labeling electron microscopy of ultrathin cryosections revealed that CRISP2 was present in the perinuclear theca and neck region of the sperm head, as well as in the outer dense fibers and the fibrous sheath of the sperm tail. Interestingly, we found that under native, non-reducing conditions CRISP2 formed oligomers both in the tail and the head but with different molecular weights and different biochemical properties. The tail oligomers were insensitive to reducing conditions but nearly complete dissociated into monomers under 8 M urea treatment, while the head 250 kDa CRISP2 positive oligomer completely dissociated into CRISP2 monomers under reducing conditions. The head specific dissociation of CRISP2 oligomer is likely a result of the reduction of various sulfhydryl groups in the cysteine rich domain of this protein. The sperm head CRISP2 shared typical solubilization characteristics with other perinuclear theca proteins as was shown with sequential detergent and salt treatments. Thus, CRISP2 is likely to participate in the formation of functional protein complexes in both the sperm tail and sperm head, but with differing oligomeric organization and biochemical properties. Future studies will be devoted to the understand the role of CRISP2 in sperm protein complexes formation and how this contributes to the fertilization processes.

The nitric oxide donor sodium nitroprusside inhibits mineralization in ATDC5 cells.

The aim of this study was to test whether the nitric oxide (NO) donor sodium nitroprusside (SNP) has an effect on mineralization in ATDC5 cells. Mineralization in ATDC5 cell culture was induced by addition of beta-glycerophosphate or inorganic phosphate, visualized by staining precipitated calcium with an alizarin red stain, and quantified using atomic absorption spectrometry. SNP was shown to inhibit the mineralization of ADTC5 cells. This inhibition was not affected by inhibitors of guanylyl cyclase nor mimicked by a cyclic guanosine monophosphate (cGMP) analog. Furthermore, SNP did not inhibit phosphate uptake or inhibit apoptosis in ATDC5 cells. These findings indicate that SNP can specifically inhibit matrix mineralization via a cGMP-independent pathway and that the effect is not mediated by inhibition of phosphate transport or apoptosis. These results suggest a preventive role of NO in premature or pathological mineralization.

Effects of methyl-beta-cyclodextrin-mediated cholesterol depletion in porcine sperm compared to somatic cells.

In this study, the use of methyl-beta-cyclodextrin (MBCD) to support capacitation of sperm cells was studied. Sperm were incubated with MBCD or alternatively capacitated in an in vitro fertilization medium. The effects of these incubations on phospholipid scrambling (using merocyanin), cholesterol depletion, GM-1 localization (using cholera-toxin B (CTX)), and membrane deterioration were assessed. For comparison, this was also tested in MBCD-treated MDCK cells. In MDCK cells, upto 71% of cholesterol was depleted, which coincided with a more diffuse CTX staining without any obvious effects on cell viability. In sperm, a similar depletion of 53% cholesterol was found after a 10 mM MBCD treatment. However, no merocyanin response was observed in viable sperm after MBCD treatments (indicating a lack of membrane changes associated with sperm capacitation). In contrast to MDCK, cells >1 mM MBCD caused plasma membrane disintegration and rendered sperm immotile. At higher concentrations also acrosome disruption was noted. CTX staining was absent at < 0.1 mM MBCD incubations but appeared at higher MBCD levels and was found to be specific for deteriorated cells that showed morphological signs of acrosome disruption. No significant plasma membrane deterioration, acrosome disruption, and sperm immotility nor CTX staining and only a modest (< 15%) cholesterol depletion were observed in conventionally capacitated sperm, where 40% of the intact sperm showed merocyanin staining. Taken together, the results indicate that membranes of sperm are more sensitive to MBCD-mediated cholesterol depletion than MDCK cells and that the use of MBCD to support sperm capacitation cannot be recommended due to its spermicidal effects.

Capacitation-dependent concentration of lipid rafts in the apical ridge head area of porcine sperm cells.

Lipid architecture of the plasma membrane plays an important role in the capacitation process of the sperm cell. During this process, an increase in membrane fluidity takes place, which coincides with a redistribution of cholesterol to the apical region of the head plasma membrane and subsequently an efflux of cholesterol. Cholesterol is also a major player in the formation of lipid rafts or microdomains in the membrane. Lipid rafts favour specific protein-protein interactions by concentrating certain proteins in these microdomains while excluding others. In this study, we investigated the organization of lipid rafts during in vitro capacitation of boar sperm cells. We report on the presence of the lipid raft-specific proteins caveolin-1 and flotillin-1 in sperm cells. Capacitation induced a change in membrane distribution of these proteins. Lipid analysis on detergent-resistant membranes (DRMs) of sperm cells indicated that capacitation induces a lipid raft concentration rather than a disintegration of lipid rafts, because the total amount of lipid in the DRM fraction remained unaltered. Using a proteomic approach, we identified several major DRM proteins, including proteins involved in capacitation-dependent processes and zona pellucida binding. Our data indicate that sperm raft reorganization may facilitate capacitation-specific signalling events and binding to the zona pellucida.

Recruitment to Golgi membranes of ADP-ribosylation factor 1 is mediated by the cytoplasmic domain of p23.

Binding to Golgi membranes of ADP ribosylation factor 1 (ARF1) is the first event in the initiation of COPI coat assembly. Based on binding studies, a proteinaceous receptor has been proposed to be critical for this process. We now report that p23, a member of the p24 family of Golgi-resident transmembrane proteins, is involved in ARF1 binding to membranes. Using a cross-link approach based on a photolabile peptide corresponding to the cytoplasmic domain of p23, the GDP form of ARF1 (ARF1-GDP) is shown to interact with p23 whereas ARF1-GTP has no detectable affinity to p23. The p23 binding is shown to localize specifically to a 22 amino acid C-terminal fragment of ARF1. While a monomeric form of a non-photolabile p23 peptide does not significantly inhibit formation of the cross-link product, the corresponding dimeric form does compete efficiently for this interaction. Consistently, the dimeric p23 peptide strongly inhibits ARF1 binding to native Golgi membranes suggesting that an oligomeric form of p23 acts as a receptor for ARF1 before nucleotide exchange takes place.

Sphingomyelin-enriched microdomains at the Golgi complex.

Sphingomyelin- and cholesterol-enriched microdomains can be isolated as detergent-resistant membranes from total cell extracts (total-DRM). It is generally believed that this total-DRM represents microdomains of the plasma membrane. Here we describe the purification and detailed characterization of microdomains from Golgi membranes. These Golgi-derived detergent-insoluble complexes (GICs) have a low buoyant density and are highly enriched in lipids, containing 25% of total Golgi phospholipids including 67% of Golgi-derived sphingomyelin, and 43% of Golgi-derived cholesterol. In contrast to total-DRM, GICs contain only 10 major proteins, present in nearly stoichiometric amounts, including the alpha- and beta-subunits of heterotrimeric G proteins, flotillin-1, caveolin, and subunits of the vacuolar ATPase. Morphological data show a brefeldin A-sensitive and temperature-sensitive localization to the Golgi complex. Strikingly, the stability of GICs does not depend on its membrane environment, because, after addition of brefeldin A to cells, GICs can be isolated from a fused Golgi-endoplasmic reticulum organelle. This indicates that GIC microdomains are not in a dynamic equilibrium with neighboring membrane proteins and lipids. After disruption of the microdomains by cholesterol extraction with cyclodextrin, a subcomplex of several GIC proteins including the B-subunit of the vacuolar ATPase, flotillin-1, caveolin, and p17 could still be isolated by immunoprecipitation. This indicates that several of the identified GIC proteins localize to the same microdomains and that the microdomain scaffold is not required for protein interactions between these GIC proteins but instead might modulate their affinity.

Evidence for segregation of sphingomyelin and cholesterol during formation of COPI-coated vesicles.

In higher eukaryotes, phospholipid and cholesterol synthesis occurs mainly in the endoplasmic reticulum, whereas sphingomyelin and higher glycosphingolipids are synthesized in the Golgi apparatus. Lipids like cholesterol and sphingomyelin are gradually enriched along the secretory pathway, with their highest concentration at the plasma membrane. How a cell succeeds in maintaining organelle-specific lipid compositions, despite a steady flow of incoming and outgoing transport carriers along the secretory pathway, is not yet clear. Transport and sorting along the secretory pathway of both proteins and most lipids are thought to be mediated by vesicular transport, with coat protein I (COPI) vesicles operating in the early secretory pathway. Although the protein constituents of these transport intermediates are characterized in great detail, much less is known about their lipid content. Using nano-electrospray ionization tandem mass spectrometry for quantitative lipid analysis of COPI-coated vesicles and their parental Golgi membranes, we find only low amounts of sphingomyelin and cholesterol in COPI-coated vesicles compared with their donor Golgi membranes, providing evidence for a significant segregation from COPI vesicles of these lipids. In addition, our data indicate a sorting of individual sphingomyelin molecular species. The possible molecular mechanisms underlying this segregation, as well as implications on COPI function, are discussed.

Site-specific photocrosslinking to probe interactions of Arf1 with proteins involved in budding of COPI vesicles.

ADP-ribosylation factor 1 (Arf1) plays an important role in early and intra-Golgi protein trafficking. During this process, Arf1 interacts with many different proteins and other molecules that regulate its state of activation or are involved in its intracellular function. To determine which of these proteins interact directly with Arf1 during coat protein type I (COPI) vesicle biogenesis, we probed the molecular environment of Arf1 by use of site-specific photocrosslinking. This method was first used successfully in the field of protein trafficking to study the mechanisms involved in protein translocation across the endoplasmic reticulum during protein synthesis. In such a hydrophobic environment, crosslink yields of up to 30% have been observed. We have now applied this method to study the mechanism of vesicle budding from the cytosolic face of the Golgi apparatus, an aqueous environment. Although the crosslink yield is significantly lower under these conditions, due to predominant reaction of the photolabile probes with water, a specific interaction of Arf1 with subunits of coatomer, the major coat protein of COPI vesicles, could readily be identified.

GTP-dependent binding of ADP-ribosylation factor to coatomer in close proximity to the binding site for dilysine retrieval motifs and p23.

A site-directed photocross-linking approach was employed to determine components that act downstream of ADP-ribosylation factor (ARF). To this end, a photolabile phenylalanine analog was incorporated at various positions of the putative effector region of the ARF molecule. Depending on the position of incorporation, we find specific and GTP-dependent interactions of ARF with two subunits of the coatomer complex, beta-COP and gamma-COP, as well as an interaction with a cytosolic protein (approximately 185 kDa). In addition, we observe homodimer formation of ARF molecules at the Golgi membrane. These data suggest that the binding site of ARF to coatomer is at the interface of its beta- and gamma-subunits, and this is in close proximity to the second site of interaction of coatomer with the Golgi membrane, the binding site within gamma-COP for cytosolic dibasic/diphenylalanine motifs.

p24 and p23, the major transmembrane proteins of COPI-coated transport vesicles, form hetero-oligomeric complexes and cycle between the organelles of the early secretory pathway.

COPI-coated vesicles that bud off the Golgi complex contain two major transmembrane proteins, p23 and p24. We have localized the protein at the Golgi complex and at COPI-coated vesicles. Transport from the intermediate compartment (IC) to the Golgi can be blocked at 15 degrees C, and under these conditions p24 accumulates in peripheral punctated structures identified as IC. Release from the temperature block leads to a redistribution of p24 to the Golgi, showing that p24, similar to p23, cycles between the IC and Golgi complex. Immunoprecipitations of p24 from cell lysates and from detergent-solubilized Golgi membranes and COPI-coated vesicles show that p24 and p23 interact with each other to form a complex. Transient transfection of p23 in HeLa cells shows that p23 and p24 colocalize in structures induced by the overexpression of p23. Taken together p24 interacts with p23 and constitutively cycles between the organelles of the early secretory pathway.

Receptor-induced polymerization of coatomer.

Coatomer, the coat protein complex of COPI vesicles, is involved in the budding of these vesicles, but the underlying mechanism is unknown. Toward a better understanding of this process, the interaction between coatomer and the cytoplasmic domain of a major transmembrane protein of COPI vesicles, p23, was studied. Interaction of coatomer with this peptide domain results in a conformational change and polymerization of the complex in vitro. This changed conformation also is observed in vivo, i.e., on the surface of authentic, isolated COPI vesicles. An average of four peptides was found associated with one coatomer complex after polymerization. Based on these results, we propose a mechanism by which the induced conformational change of coatomer results in its polymerization, and thus drives formation of the bud on the Golgi membrane during biogenesis of a COPI vesicle.

Uptake by COPI-coated vesicles of both anterograde and retrograde cargo is inhibited by GTPgammaS in vitro.

On the basis of the cell surface protein CD8 we have constructed reporter molecules for both anterograde and retrograde transport from the Golgi complex. The cytoplasmic tail of CD8 was exchanged by a construct comprising a hemagglutinin (HA) epitope, the C-terminal sequence of the viral protein E19 (containing a KKXX retrieval signal) followed by a myc epitope (CD8-LT). Due to this masking of the KKXX retrieval signal CD8-LT is transported to the cell surface. Since the KKXX motif is joined to the myc epitope via a thrombin cleavage site, CD8-LT in isolated Golgi membranes can be proteolytically converted into an unmasked reporter molecule for retrograde transport (CD8-ST) in vitro. A CHO cell line stably expressing CD8-LT was generated and used for the isolation of Golgi membranes. These membranes were shown to contain CD8-LT en route to the cell surface. By addition of thrombin, CD8-LT could be efficiently converted into CD8-ST, and this allows us to study the sorting into coat protein COPI-coated vesicles of these different kinds of cargo on a comparative basis. COPI-coated vesicles were generated in vitro from Golgi membranes containing either CD8-LT or CD8-ST. When the incubation was performed in the presence of GTP, both CD8-LT and CD8-ST were packaged into COPI-coated vesicles. However, COPI-coated vesicles generated in the presence of the slowly hydrolyzable analogue of GTP, GTP(&ggr ;)S contained strikingly lower amounts of CD8-LT and CD8-ST. While COPI-coated vesicles accumulated about 12-fold in the presence of GTPgammaS these vesicles together contained only one fifth of cargo compared to the few vesicles generated in the absence of GTPgammaS. These data indicate that cargo packaging into COPI-coated vesicles requires hydrolysis of GTP.

A putative heterotrimeric G protein inhibits the fusion of COPI-coated vesicles. Segregation of heterotrimeric G proteins from COPI-coated vesicles.

Heterotrimeric G proteins have been implicated in the regulation of intracellular protein transport, but their mechanism of action remains unclear. In vivo, secretion of chromogranin B, tagged with the green fluorescent protein, was inhibited by the addition of a general activator of trimeric G proteins (AlF4-) to stably transfected Vero cells and resulted in an accumulation of the tagged protein in the Golgi apparatus. In an in vitro assay that reconstitutes intra-Golgi protein transport, we find that a membrane-bound and AlF4--sensitive factor is involved in the fusion reaction. To determine whether this effect is mediated by a heterotrimeric G protein localized to COPI-coated transport vesicles, we determined the presence of G proteins on these vesicles and found that they were segregated relative to the donor membranes. Because G proteins do not have an obvious sorting, retention, or retrieval signal, we considered the possibility that other interactions might be responsible for this segregation. In agreement with this, we found that trimeric G proteins from isolated Golgi membranes were partially insoluble in Triton X-100. Identification of the proteins that interact with the heterotrimeric G proteins in the Golgi-derived detergent-insoluble complex might help to reveal the regulation of protein secretion mediated by heterotrimeric G proteins.

Direct and GTP-dependent interaction of ADP ribosylation factor 1 with coatomer subunit beta.

A site-directed photocrosslink approach was used to elucidate components that interact directly with ADP- ribosylation factor (ARF)-GTP during coat assembly. Two ARF mutants were generated that contain a photolabile amino acid at positions distant to each other within the ARF molecule. Here we show that one of the two positions specifically interacts with coatomer subunit beta both on Golgi membranes and in isolated coat protein complex type I (COPI)-coated vesicles. Thus, a direct and GTP-dependent interaction of coatomer via beta-coat protein complex (COP) with ARF is involved in the coating of COPI-coated vesicles. These data implicate a bivalent interaction of the complex with the donor membrane during vesicle formation.

Regulated exocytosis in chromaffin cells. A potential role for a secretory granule-associated ARF6 protein.

The ADP-ribosylation factor (ARF) GTP-binding proteins are believed to function as regulators of vesicular budding and fusion along the secretory pathway. To investigate the role of ARF in regulated exocytosis, we have examined its intracellular distribution in cultured chromaffin cells by subcellular fractionation and immunoreplica analysis. We found that ARF6 is specifically associated with the membrane of purified secretory chromaffin granules. Chemical cross-linking and immunoprecipitation experiments suggested that ARF6 may be part of a complex with betagamma subunits of trimeric G proteins. Stimulation of intact chromaffin cells or direct elevation of cytosolic calcium in permeabilized cells triggered the rapid dissociation of ARF6 from secretory granules. This effect could be inhibited by AlF4- which selectively activates trimeric G proteins. Furthermore, a synthetic myristoylated peptide corresponding to the N-terminal domain of ARF6 strongly inhibited calcium-evoked secretion in streptolysin-O-permeabilized chromaffin cells. The possibility that ARF6 plays a role in the effector pathway by which trimeric G proteins control exocytosis in chromaffin cells is discussed.

A major transmembrane protein of Golgi-derived COPI-coated vesicles involved in coatomer binding.

Formation of non-clathrin-coated vesicles requires the recruitment of several cytosolic factors to the Golgi membrane. To identify membrane proteins involved in this budding process, a highly abundant type I transmembrane protein (p23) was isolated from mammalian Golgi-derived COPI-coated vesicles, and its cDNA was cloned and sequenced. It belongs to the p24 family of proteins involved in the budding of transport vesicles (Stamnes, M.A., M.W. Craighead, M.H. Hoe, N. Lampen, S. Geromanos, P. Tempst, and J.E. Rothman. 1995. Proc. Natl. Acad. Sci. USA. 92:8011-8015). p23 consists of a large NH2-terminal luminal domain and a short COOH-terminal cytoplasmic tail (-LRRFFKAKKLIE-CO2-) that shows similarity, but not identity, with the sequence motif-KKXX-CO2-, known as a signal for retrieval of escaped ER-resident membrane proteins (Jackson, M.R., T. Nilsson, and P.A. Peterson. 1990. EMBO (Eur. Mol. Biol. Organ.) J. 9:3153-3162; Nilsson, T., M. Jackson, and P.A. Peterson. 1989. Cell. 58:707-718). The cytoplasmic tail of p23 binds to coatomer with similar efficiency as known KKXX motifs. However, the p23 tail differs from the KKXX motif in having an additional motif needed for binding of coatomer. p23 is localized to Golgi cisternae and, during vesicle formation, it concentrates into COPI-coated buds and vesicles. Biochemical analysis revealed that p23 is enriched in vesicles by a factor of approximately 20, as compared with the donor Golgi fraction, and is present in amounts stoichiometric to the small GTP-binding protein ADP-ribosylation factor (ARF) and coatomer. From these data we conclude that p23 represents a Golgi-specific receptor for coatomer involved in the formation of COPI-coated vesicles.

Forskolin stimulates detoxification of brefeldin A.

Forskolin has been shown to prevent the effects brefeldin A (BFA) exerts on many mammalian cells with respect to the disassembly of the Golgi apparatus as well as an increase of sphingomyelin synthesis (Lippincott, S. J., Glickman, J., Donaldson, J. G., Robbins, J., Kreis, T. E., Seamon, K. B., Sheetz, M. P., and Klausner, R. D. (1991) J. Cell Biol. 112, 567-577). It has been speculated that forskolin interferes with the action of BFA by competition for the binding of BFA to its target protein, which is most likely the Golgi-localized nucleotide exchange factor specific for ADP-ribosylation factor 1. Here we show that in vitro forskolin does not prevent inhibition of Golgi-catalyzed nucleotide exchange by BFA. Therefore it appears unlikely that forskolin and BFA bind to the same target protein. Using [3H]BFA we have measured detoxification of BFA by Chinese hamster ovary (CHO) cells. BFA is secreted from CHO cells as cysteine and glutathione conjugates (Brüning, A., Ishikawa, T., Kneusel, R. E., Matern, U., Lottspeich, F., and Wieland, F. T. (1992) J. Biol. Chem. 267, 7726-7732). We present evidence that forskolin treatment of CHO cells results in increased levels of Cys-BFA, the major BFA conjugate secreted by CHO cells, in the medium. Elevated levels of Cys-BFA are also found intracellularly. The effect of forskolin is shown to be independent of its ability to raise the intracellular concentration of cyclic AMP. Therefore, we suggest that the effect of forskolin on BFA-induced disassembly of the Golgi apparatus might be due to an enhanced detoxification of the drug.

Role of heterotrimeric GTP binding proteins in vesicular protein transport: indications for both classical and alternative G protein cycles.

Heterotrimeric G proteins are involved in hormonal signal transduction across the plasma membrane. Recent evidence suggests that they have a role in vesicular protein transport as well. Biochemical probes that interfere with the classical G protein cycle have been applied to the field of intracellular membrane transport to study their mechanism of action. Evidence has been obtained that intracellular G proteins act both through classical and alternative G protein cycles.

Hydrolysis of bound GTP by ARF protein triggers uncoating of Golgi-derived COP-coated vesicles.

The cycle of nucleotide exchange and hydrolysis by a small GTP-binding protein, ADP-ribosylation factor (ARF), helps to provide vectoriality to vesicle transport. Coat assembly is triggered when ARF binds GTP, initiating transport vesicle budding, and coat disassembly is triggered when ARF hydrolyzes GTP, allowing the uncoated vesicle to fuse.

Binding of coatomer to Golgi membranes requires ADP-ribosylation factor.

Coatomer, a complex of seven proteins, appears to be the precursor of the coat structure of non-clathrin-coated Golgi-derived vesicles. Another component of this vesicle coat is the cytosolic protein ADP-ribosylation factor (ARF). Like coatomer, ARF appears to reversibly associate with Golgi membranes. We now report that ARF is required for coatomer binding to Golgi membranes and that myristoylated, but not non-myristoylated, ARF is the required species. We utilize an antibody directed against the beta-subunit of coatomer (beta-COP) to follow coatomer binding. ARF and beta-COP bind stoichiometrically to Golgi membranes. ARF-dependent beta-COP binding requires a membrane-associated protein, is saturable, and is enhanced in the presence of stable GTP analogues like guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). ARF and beta-COP bind sequentially to Golgi membranes, since beta-COP can be bound to reisolated membranes that had been previously incubated with ARF and GTP gamma S. We conclude that membrane-bound ARF confers to Golgi membranes all of the requirements for specific beta-COP binding.