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.

Two distinct populations of ARF bound to Golgi membranes.

ADP-ribosylation factor (ARF) is a small molecular weight GTP-binding protein (20 kD) and has been implicated in vesicular protein transport. The guanine nucleotide, bound to ARF protein is believed to modulate the activity of ARF but the mechanism of action remains elusive. We have previously reported that ARF binds to Golgi membranes after Brefeldin A-sensitive nucleotide exchange of ARF-bound GDP for GTP gamma S. Here we report that treatment with phosphatidylcholine liposomes effectively removed 40-60% of ARF bound to Golgi membranes with nonhydrolyzable GTP, presumably by competing for binding of activated ARF to lipid bilayers. This revealed the presence of two different pools of ARF on Golgi membranes. Whereas total ARF binding did not appear to be saturable, the liposome-resistant pool is saturable suggesting that this pool of ARF is stabilized by interaction with a Golgi membrane-component. We propose that activation of ARF by a guanine nucleotide-exchange protein results in association of myristoylated ARF GTP with the lipid bilayer of the Golgi apparatus. Once associated with the membrane, activated ARF can diffuse freely to associate stably with a target protein or possibly can be inactivated by a GTPase activating protein (GAP) activity.

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.

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.

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.

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.

Purification of nonspecific lipid transfer protein (sterol carrier protein 2) from human liver and its deficiency in livers from patients with cerebro-hepato-renal (Zellweger) syndrome.

The nonspecific lipid transfer protein (i.e., sterol carrier protein 2) from human liver was purified to homogeneity using ammonium sulfate precipitation, CM-cellulose chromatography, molecular sieve chromatography and fast protein liquid chromatography. Its amino acid composition was determined and found to be very similar to that of the nonspecific lipid transfer protein from bovine and rat liver with, as main feature, the absence of arginine, histidine and tyrosine. By way of a specific enzyme immunoassay using affinity-purified antibodies, the levels of nonspecific lipid transfer protein were determined in human livers. Levels varied from approximately 150 ng nonspecific lipid transfer protein per mg 105,000 X g supernatant protein for juvenile and adult humans to 40 ng per mg supernatant protein for a young infant. Levels of nonspecific lipid transfer protein in livers of infants with cerebro-hepato-renal (Zellweger) syndrome were extremely low (i.e., 2 ng per mg supernatant protein). Immunoblotting revealed the presence of crossreactive proteins of molecular masses of 40,000 and 58,000. The 40 kDa and 58 kDa proteins occurred in control livers, whereas only the 40 kDa protein was present in Zellweger livers. As in rat the 58 kDa protein could be demonstrated in a peroxisomal preparation isolated from an adult liver. A possible link between the occurrence of nonspecific lipid transfer protein and the presence of peroxisomes is discussed.

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.

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.

Racial differences in patients with adenocarcinoma of the endometrium.

OBJECTIVE: To determine the differences between white and black women with regard to the presentation and behavior of adenocarcinoma of the endometrium. METHOD: Records of 273 (68%) white patients and 117 (32%) black patients with endometrial adenocarcinoma were reviewed in Bloemfontein, South Africa. Survival data was calculated according to the direct method where losses in follow-up were regarded as tumor deaths. RESULTS: Most patients (82%) were treated by pre-operative radium followed by total abdominal hysterectomy and bilateral salpingo-oophorectomy, with post-operative external irradiation where indicated. Pre-operatively, fewer black women had reached FIGO stage I, while a larger number had advanced to stages II-IV (P = 0.0024). In addition, the tumor differentiation was more often poor in the black group (P < 0.0001). Ten-year follow-up was achieved in 84% of the white patients and 51% of the black patients and the 10-year survival figures were 67% for white patients and 28% for blacks (P < 0.0001). CONCLUSION: Endometrial adenocarcinoma is a more aggressive disease in black women than it is in whites.

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.

Synthesis of beta-hematin by dehydrohalogenation of hemin.

beta-Hematin, the metalloporphyrin coordination polymer present in malaria pigment, is prepared in high yield by the abstraction of HCl from hemin with a non-coordinating base in strictly anhydrous conditions. The product of this preparation is characterized by elemental analysis, infrared and diffuse reflectance spectroscopies and demonstrated to be identical to prior preparations.

Inhibition by brefeldin A of a Golgi membrane enzyme that catalyses exchange of guanine nucleotide bound to ARF.

A wide variety of membrane transformations important in intracellular transport are inhibited by the fungal metabolite brefeldin A (refs 1-4), implying that the target for this drug is central to the formation and maintenance of subcellular compartments. Brefeldin A added to cells causes the rapid and reversible dissociation of a Golgi-associated peripheral membrane protein (M(r) 110,000) which was found to be identical to one of the subunits of the coat of Golgi-derived (non-clathrin) coated vesicles, beta-COP, implying that brefeldin A prevents transport by blocking the assembly of coats and thus the budding of enclosed vesicles. In addition to the coatomer (a cytosol-derived complex of seven polypeptide chains, one of which is beta-COP), the non-clathrin (COP) coat of Golgi-derived vesicles contains stoichiometric amounts of a small (M(r) approximately 20,000) GTP-binding protein, the ADP-ribosylation factor (ARF). Binding of ARF to Golgi membranes is necessary before coatomer/beta-COP can bind these membranes (ref. 12; and D. J. Palmer et al., manuscript submitted), so the primary effect of brefeldin A seems to be on the reaction responsible for ARF binding. Indeed, like beta-COP, ARF is dissociated from the Golgi complex by treatment with brefeldin A and brefeldin A prevents ARF from associating in vitro, but the mechanism of this action by brefeldin A has been unclear. Here we report the discovery of an enzyme in a Golgi-enriched fraction that catalyses guanine nucleotide (GDP-GTP) exchange on ARF-1 protein, and which is inhibited by brefeldin A. We suggest that activation of ARF proteins for membrane localization by compartmentalized exchange enzymes is in general the first committed step in membrane transformation pathways.

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.

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.

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.

Reconstitution of steps in the constitutive secretory pathway in permeabilized cells. Secretion of glycosylated tripeptide and truncated sphingomyelin.

The constitutive secretory pathway has been reconstituted in mechanically permeabilized Chinese hamster ovary cells using two secretory markers, an acyltripeptide (N-octanoyl-Asn-Tyr-Thr-NH2) that is glycosylated at Asn in the endoplasmic reticulum and a truncated ceramide that is converted to sphingomyelin. Secretion of these bulk phase markers is dependent on cytosolic proteins and ATP. Secretion of both the glycosylated tripeptide and truncated sphingomyelin was inhibited at 15 degrees C. These results are taken as evidence that the vesicle flow to the plasma membrane (rather than artificial lysis of endoplasmic reticulum or Golgi cisternae) is required for the release of markers to the medium. Guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), a nonhydrolyzable analogue of GTP, inhibited secretion, resulting in an accumulation of both the glycosylated tripeptide and truncated sphingomyelin in the semi-intact cell. Inhibition of secretion by GTP gamma S was not observed in the presence of the aminoglycoside antibiotic neomycin.