Quantitative immunogold localization of dipeptidyl peptidase IV (DPP IV) in rat liver cells

We investigated ultrastructural localization of dipeptidyl peptidase IV (DPP IV) [EC3.4.14 5] in rat liver cells quantitatively by post embedding protein A-gold technique. In the hepatocyte, DPP IV was mainly localized on the bile canalicular surface and the lysosomal membranes, but were scarcely detectable on the sinusoid-lateral surface. A small number of DPP IV was also detected in the trans region of the Golgi apparatus, suggesting that this part may play important roles in intracellular transport or recycling of this enzyme. In the endothelial cell, DPP IV existed on the whole surface of the plasma membrane and the lysosomes. In the Kupffer cell DPP IV was mainly localized in lysosomes and a few were detected on the plasma membrane.     

Newly synthesized canalicular ABC transporters are directly targeted from the Golgi to the hepatocyte apical domain in rat liver

Newly synthesized canalicular ectoenzymes and a cell adhesion molecule (cCAM105) have been shown to traffic from the Golgi to the basolateral plasma membrane, from where they transcytose to the apical bile canalicular domain. It has been proposed that all canalicular proteins are targeted via this indirect route in hepatocytes. We studied the membrane targeting of rat canalicular proteins by in vivo [(35)S]methionine metabolic labeling followed by preparation of highly purified Golgi membranes and canalicular (CMVs) and sinusoidal/basolateral (SMVs) membrane vesicles and subsequent immunoprecipitation. In particular, we compared membrane targeting of newly synthesized canalicular ABC (ATP-binding cassette) transporters MDR1, MDR2, and SPGP (sister of P-glycoprotein) with that of cCAM105. Significant differences were observed in metabolic pulse-chase labeling experiments with regard to membrane targeting of these apical proteins. After a chase time of 15 min, cCAM105 appeared exclusively in SMVs, peaked at 1 h, and progressively declined thereafter. In CMVs, cCAM105 was first detected after 1 h and subsequently increased for 3 h. This findings confirm the transcytotic targeting of cCAM105 reported in earlier studies. In contrast, at no time point investigated were MDR1, MDR2, and SPGP detected in SMVs. In CMVs, MDR1 and MDR2 appeared after 30 min, whereas SPGP appeared after 2 h of labeling. In Golgi membranes, each of the ABC transporters peaked at 30 min and was virtually absent thereafter. These data suggest rapid, direct targeting of newly synthesized MDR1 and MDR2 from the Golgi to the bile canaliculus and transient sequestering of SPGP in an intracellular pool en route from the Golgi to the apical plasma membrane. This study provides biochemical evidence for direct targeting of newly synthesized apical ABC transporters from the Golgi to the bile canaliculus in vivo.     

Identification of HAX-1 as a protein that binds bile salt export protein and regulates its abundance in the apical membrane of Madin-Darby canine kidney cells

ATP-binding cassette (ABC)-type proteins are essential for bile formation in vertebrate liver. BSEP, MDR1, MDR2, and MRP2 ABC transporters are targeted to the apical (canalicular) membrane of hepatocytes where they execute ATP-dependent transport of bile acids, drugs, amphipathic cations, phospholipids, and conjugated organic anions, respectively. Changes in activity and abundance of transporters in the canalicular membrane regulate bile flow; however, little is known regarding cellular proteins that bind ABC transporters and regulate their trafficking. A yeast two-hybrid screen identified HAX-1 as a binding partner for BSEP, MDR1, and MDR2. The interactions were validated biochemically by glutathione S-transferase pull-down and co-immunoprecipitation assays. BSEP and HAX-1 were over-represented in rat liver subcellular fractions enriched for canalicular membrane vesicles, microsomes, and clathrin-coated vesicles. HAX-1 was bound to BSEP, MDR1, and MDR2 in canalicular membrane vesicles and co-localized with BSEP and MDR1 in the apical membrane of Madin-Darby canine kidney (MDCK) cells. RNA interference of HAX-1 increased BSEP levels in the apical membrane of MDCK cells by 71%. Pulse-chase studies indicated that HAX-1 depletion did not affect BSEP translation, post-translational modification, delivery to the plasma membrane, or half-life. HAX-1 depletion resulted in an increased peak of metabolically labeled apical membrane BSEP at 4 h and enhanced retention at 6 and 9 h. HAX-1 also interacts with cortactin. Expression of dominant negative cortactin increased steady state levels of BSEP 2-fold in the apical membrane of MDCK cells, as did expression of dominant negative EPS15. These findings suggest that HAX-1 and cortactin participate in BSEP internalization from the apical membrane.     

AKAP350 Is involved in the development of apical "canalicular" structures in hepatic cells HepG2

Hepatocytes are epithelial cells whose apical poles constitute the bile canaliculi. The establishment and maintenance of canalicular poles is a finely regulated process that dictates the efficiency of primary bile secretion. Protein kinase A (PKA) modulates this process at different levels. AKAP350 is an A-kinase anchoring protein that scaffolds protein complexes involved in modulating the dynamic structures of the Golgi apparatus and microtubule cytoskeleton, facilitating microtubule nucleation at this organelle. In this study, we evaluated whether AKAP350 is involved in the development of bile canaliculi-like structures in hepatocyte derived HepG2 cells. We found that AKAP350 recruits PKA to the centrosomes and Golgi apparatus in HepG2 cells. De-localization of AKAP350 from these organelles led to reduced apical cell polarization. A decrease in AKAP350 expression inhibited the formation of canalicular structures and impaired F-actin organization at canalicular poles. Furthermore, loss of AKAP350 expression led to diminished polarized expression of the p-glycoprotein (MDR1/ABCB1) at the apical "canalicular" membrane. AKAP350 knock down effects on canalicular structures formation and actin organization could be mimicked by inhibition of Golgi microtubule nucleation by depletion of CLIP associated proteins (CLASPs). Our data reveal that AKAP350 participates in mechanisms which determine the development of canalicular structures as well as accurate canalicular expression of distinct proteins and actin organization, and provide evidence on the involvement of Golgi microtubule nucleation in hepatocyte apical polarization.     

Transporters on demand: intrahepatic pools of canalicular ATP binding cassette transporters in rat liver

ABC transporter trafficking in rat liver induced by cAMP or taurocholate and [(35)S]methionine metabolic labeling followed by subcellular fractionation were used to identify and characterize intrahepatic pools of ABC transporters. ABC transporter trafficking induced by cAMP or taurocholate is a physiologic response to a temporal demand for increased bile secretion. Administration of cAMP or taurocholate to rats increased amounts of SPGP, MDR1, and MDR2 in the bile canalicular membrane by 3-fold; these effects abated after 6 h and were insensitive to prior treatment of rats with cycloheximide. Half-lives of ABC transporters were 5 days, which suggests cycling of ABC transporters between canalicular membrane and intrahepatic sites before degradation. In vivo [(35)S]methionine labeling of rats followed by immunoprecipitation of (sister of P-glycoprotein) (SPGP) from subcellular liver fractions revealed a steady state distribution after 20 h of SPGP between canalicular membrane and a combined endosomal fraction. After mobilization of transporters from intrahepatic sites with cAMP or taurocholate, a significant increase in the amount of ABC transporters in canalicular membrane vesicles was observed, whereas the decrease in the combined endosomal fraction remained below detection limits in Western blots. This observation is in accordance with relatively large intracellular ABC transporter pools compared with the amount present in the bile canalicular membrane. Furthermore, trafficking of newly synthesized SPGP through intrahepatic sites was accelerated by additional administration of cAMP but not by taurocholate, indicating two distinct intrahepatic pools. Our data indicate that ABC transporters cycle between the bile canaliculus and at least two large intrahepatic ABC transporter pools, one of which is mobilized to the canalicular membrane by cAMP and the other, by taurocholate. In parallel to regulation of other membrane transporters, we propose that the "cAMP-pool" in hepatocytes corresponds to a recycling endosome, whereas recruitment from the "taurocholate-pool" involves a hepatocyte-specific mechanism.     

Myosin II regulatory light chain is required for trafficking of bile salt export protein to the apical membrane in Madin-Darby canine kidney cells

BSEP, MDR1, and MDR2 ATP binding cassette transporters are targeted to the apical (canalicular) membrane of hepatocytes, where they mediate ATP-dependent secretion of bile acids, drugs, and phospholipids, respectively. Sorting to the apical membrane is essential for transporter function; however, little is known regarding cellular proteins that bind ATP binding cassette proteins and regulate their trafficking. A yeast two-hybrid screen of a rat liver cDNA library identified the myosin II regulatory light chain, MLC2, as a binding partner for BSEP, MDR1, and MDR2. The interactions were confirmed by glutathione S-transferase pulldown and co-immunoprecipitation assays. BSEP and MLC2 were overrepresented in a rat liver subcellular fraction enriched in canalicular membrane vesicles, and MLC2 colocalized with BSEP in the apical domain of hepatocytes and polarized WifB, HepG2, and Madin-Darby canine kidney cells. Expression of a dominant negative, non-phosphorylatable MLC2 mutant reduced steady state BSEP levels in the apical domain of polarized Madin-Darby canine kidney cells. Pulse-chase studies revealed that Blebbistatin, a specific myosin II inhibitor, severely impaired delivery of newly synthesized BSEP to the apical surface. These findings indicate that myosin II is required for BSEP trafficking to the apical membrane.     

Glucosylceramide synthesis inhibitors block pharmacologically induced dispersal of the Golgi and anterograde membrane flow from the endoplasmic reticulum: implication of sphingolipid metabolism in maintenance of the Golgi architecture and anterograde membrane flow

PDMP (D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol) and PPMP (D,L-threo-1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol), inhibitors of glucosylceramide synthesis, blocked brefeldin A (BFA)- and nordihydroguaiaretic acid-induced dispersal of the Golgi and trans Golgi network, and Golgi-derived vesicles were retained in the juxtanuclear region. PDMP and PPMP did not stabilize microtubules but blocked nocodazole-induced extensive fragmentation and dispersal of the Golgi, and large Golgi vesicles were retained in the juxtanuclear region. PPMP is a stronger inhibitor of glucosylceramide synthesis than PDMP, but PDMP showed a stronger activity against BFA-induced retrograde membrane flow. However, PPMP showed a stronger activity for Golgi disruption and inhibition of anterograde trafficking from the endoplasmic reticulum, and rebuilding of the Golgi architecture. Cumulatively, these results suggest that sphingolipid metabolism is implicated in maintenance of the Golgi architecture and anterograde membrane flow from the endoplasmic reticulum but not in Golgi dispersal induced by BFA.     

Segregation of Glucosylceramide and Sphingomyelin Occurs in the Apical to Basolateral Transcytotic Route in HepG2 Cells

HepG2 cells are highly differentiated hepatoma cells that have retained an apical, bile canalicular (BC) plasma membrane polarity. We investigated the dynamics of two BC-associated sphingolipids, glucosylceramide (GlcCer) and sphingomyelin (SM). For this, the cells were labeled with fluorescent acyl chainlabeled 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)- amino]hexanoic acid (C₆-NBD) derivatives of either GlcCer (C₆-NBD-GlcCer) or SM (C₆-NBD-SM). The pool of the fluorescent lipid analogues present in the basolateral plasma membrane domain was subsequently depleted and the apically located C₆-NBD-lipid was chased at 37°C. By using fluorescence microscopical analysis and a new assay that allows an accurate estimation of the fluorescent lipid pool in the apical membrane, qualitative and quantitative insight was obtained concerning kinetics, extent and (intra)cellular sites of the redistribution of apically located C₆-NBD-GlcCer and C₆-NBD-SM. It is demonstrated that both lipids display a preferential localization, C₆-NBD-GlcCer in the apical and C₆-NBD-SM in the basolateral area. Such a preference is expressed during transcytosis of both sphingolipids from the apical to the basolateral plasma membrane domain, a novel lipid trafficking route in HepG2 cells. Whereas the vast majority of the apically derived C₆-NBD-SM was rapidly transcytosed to the basolateral surface, most of the apically internalized C₆-NBD-GlcCer was efficiently redirected to the BC. The redirection of C₆-NBD-GlcCer did not involve trafficking via the Golgi apparatus. Evidence is provided which suggests the involvement of vesicular compartments, located subjacent to the apical plasma membrane. Interestingly, the observed difference in preferential localization of C₆-NBD-GlcCer and C₆NBD-SM was perturbed by treatment of the cells with dibutyryl cAMP, a stable cAMP analogue. While the preferential apical localization of C₆-NBD-GlcCer was amplified, dibutyryl cAMP-treatment caused apically retrieved C₆-NBD-SM to be processed via a similar pathway as that of C₆-NBD-GlcCer.

The data unambiguously demonstrate that segregation of GlcCer and SM occurs in the reverse transcytotic route, i.e., during apical to basolateral transport, which results in the preferential localization of GlcCer and SM in the apical and basolateral region of the cells, respectively. A role for non-Golgi–related, sub-apical vesicular compartments in the sorting of GlcCer and SM is proposed.

     

Effect of drugs and temperature on biosynthesis and transport of glycosphingolipids in cultured neurotumor cells

Neuroblastoma and glioma cells were grown in the presence of [³H]galactose, and the incorporation of ³H into gangliosides and the transport of newly synthesized gangliosides to the cell surface were examined under different experimental conditions. A variety of drugs, including inhibitors of protein synthesis and energy metabolism, modulators of the cytoskeleton and the ionophore monensin, had no effect on the transport of newly synthesized GD1a in neuroblastoma cells. Only low temperature effectively blocked translocation to the plasma membrane. Monensin, however, had marked effects on the biosynthesis of gangliosides and neutral glycosphingolipids. Whereas incorporation of ³H into complex glycosphingolipids was reduced, labeling of glucosylceramide was increased in cells exposed to monensin. In addition, biosynthesis of the latter glycolipid was less susceptible to low temperatures than that of more complex ones. Previous studies have implicated the Golgi apparatus as the predominant site of glycosylation of gangliosides. As monensin has been reported to interfere with the Golgi apparatus, our results indicate that glucosylceramide may be synthesized at a site that is separate from the site where further glycosylation occurs. Once synthesis of a ganglioside is completed, transport of the molecule to the cell surface proceeds under conditions of cytoskeletal disruption, energy depletion and ionic inbalance, but not low temperature.     

肝细胞极性与膜蛋白分选的分子机制

肝细胞是高度特化的极性上皮细胞,细胞质膜蛋白的分选和极性转运对于肝细胞极性的建立与维持至关重要.首先,膜蛋白在内质网中合成,随后经高尔基体加工修饰,再由反面高尔基体进一步分选,最后通过膜泡运输等不同的机制分别转运到胆汁腔面或窦状隙面,行使其特殊的功能.近些年来,细胞内负责转运的细胞器和主要的分选信号已逐步被揭示.特别是循环内体也被证明参与了胆汁腔面和窦状隙面膜蛋白的极性分选和转运.肝细胞的极性一旦遭到破坏,将会引起胆汁分泌障碍以及其他肝脏功能的损伤,从而可能导致肝脏糖脂代谢紊乱,甚至丧失正常的生理功能.因此,深入研究肝脏细胞极性的形成与维持机制,将为多种肝脏疾病的预防和治疗寻找到新的方向和靶点,具有重要的理论和临床实践意义.     

Upregulation of the expression of endogenous Mdr1 P-glycoprotein enhances lipid translocation in MDCK cells transfected with human MRP2

Various ABC transporters can translocate lipid molecules from the cytoplasmic into the exoplasmic leaflet of the plasma membrane bilayer. Two of these, MDR1 P-glycoprotein (Pgp) and MRP1, are multidrug transporters responsible for the resistance of various cancers against chemotherapy. We wanted to study whether MRP2, an ABC transporter of the bile canalicular membrane with a substrate specificity very similar to that of MRP1, is capable of translocating lipids. The translocation of short-chain lipids across the apical membrane of MDCK cells transfected with MRP2 was significantly higher than that in untransfected controls. However, the characteristics of the lipid translocation were similar to substrate transport by MDR1 and not MRP2: transport was strongly inhibited by classic MDR1 Pgp inhibitors, was independent of cellular glutathione, and was insensitive to a drug known to inhibit MRP2 activity. When tested by immunoblot, the MRP2-transfected cells expressed high levels of MRP2 but also of endogenous Mdr1. The expression of Mdr1 was unstable during maintenance of the cell line and correlated with the rate of lipid translocation across the apical membrane. We conclude that the observed increase in lipid transport in the MDCK cells transfected with MRP2 is the consequence of the upregulation of the expression of endogenous Mdr1 and that careful characterization of endogenous Mdr1 expression is needed in studies aimed to identify substrates of plasma membrane transporters.     

Glucosphingolipid dependence of hormone-stimulated inositol trisphosphate formation

The modulatory role of endogenous cellular glycosphingolipids in bradykinin-stimulated myo-inositol 1,4,5-trisphosphate (InsP3) formation by MDCK cells was evaluated utilizing the glucosylceramide synthase inhibitor, threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP). Bradykinin-stimulated InsP3 formation in intact cells and in isolated plasma membranes was significantly enhanced when cells were first depleted of their glucosphingolipids. The effect of glucosphingolipid depletion on phospholipase C activity was dependent on the duration of exposure to the inhibitor and the cellular level of glucosylceramide. Inclusion of glucosylceramide in the culture medium prevented the stimulatory effect of PDMP on InsP3 formation. It is concluded that membrane glucosphingolipids may regulate phospholipase C activity.     

Mechanisms by which cAMP increases bile acid secretion in rat liver and canalicular membrane vesicles

Bile acid secretion induced by cAMP and taurocholate is associated with recruitment of several ATP binding cassette (ABC) transporters to the canalicular membrane. Taurocholate-mediated bile acid secretion and recruitment of ABC transporters are phosphatidylinositol 3-kinase (PI3K) dependent and require an intact microtubular apparatus. We examined mechanisms involved in cAMP-mediated bile acid secretion. Bile acid secretion induced by perfusion of rat liver with dibutyryl cAMP was blocked by colchicine and wortmannin, a PI3K inhibitor. Canalicular membrane vesicles isolated from cAMP-treated rats manifested increased ATP-dependent transport of taurocholate and PI3K activity that were reduced by prior in vivo administration of colchicine or wortmannin. Addition of a PI3K lipid product, phosphoinositide 3,4-bisphosphate, but not its isomer, phosphoinositide 4,5-bisphosphate, restored ATP-dependent taurocholate in these vesicles. Addition of a decapeptide that activates PI3K to canalicular membrane vesicles increased ATP-dependent transport above baseline activity. In contrast to effects induced by taurocholate, cAMP-stimulated intracellular trafficking of the canalicular ABC transporters was unaffected by wortmannin, and recruitment of multidrug resistance protein 2, but not bile salt excretory protein (bsep), was partially decreased by colchicine. These studies indicate that trafficking of bsep and other canalicular ABC transporters to the canalicular membrane in response to cAMP is independent of PI3K activity. In addition, PI3K lipid products are required for activation of bsep in the canalicular membrane. These observations prompt revision of current concepts regarding the role of cAMP and PI3K in intracellular trafficking, regulation of canalicular bsep, and bile acid secretion.     

Glycolipid Transfer Protein Expression Is Affected by Glycosphingolipid Synthesis

Members of the glycolipid transfer protein superfamily (GLTP) are found from animals and fungi to plants and red micro-alga. Eukaryotes that encode the glucosylceramide synthase responsible for the synthesis of glucosylceramide, the precursor for most glycosphingolipids, also produce GLTPs. Cells that does not synthesize glucosylceramide neither express GLTPs. Based on this genetic relationship there must be a strong correlation between the synthesis of glucosylceramide and GLTPs. To regulate the levels of glycolipids we have used inhibitors of intracellular trafficking, glycosphingolipid synthesis and degradation, and small interfering RNA to down-regulate the activity of glucosylceramide synthase activity. We found that GLTP expression, both at the mRNA and protein levels, is elevated in cells that accumulate glucosylceramide. Monensin and brefeldin A block intracellular vesicular transport mechanisms. Brefeldin A treatment leads to accumulation of newly synthesized glucosylceramide, galactosylceramide and lactosylceramide in a fused endoplasmic reticulum-Golgi complex. On the other hand, inhibiting glycosphingolipid degradation with conduritol-B-epoxide, that generates glucosylceramide accumulation in the lysosomes, did not affect the levels of GLTP. However, glycosphingolipid synthesis inhibitors like PDMP, NB-DNJ and myriocin, all decreased glucosylceramide and GLTP below normal levels. We also found that an 80% loss of glucosylceramide due to glucosylceramide synthase knockdown resulted in a significant reduction in the expression of GLTP. We show here that interfering with membrane trafficking events and simple neutral glycosphingolipid synthesis will affect the expression of GLTP. We postulate that a change in the glucosylceramide balance causes a response in the GLTP expression, and put forward that GLTP might play a role in lipid directing and sensing of glucosylceramide at the ER-Golgi interface.     

Localization in the Golgi apparatus of rat liver UDP-Gal:glucosylceramide beta 1----4galactosyltransferase

The presence and subcellular localization of UDP-Gal:glucosylceramide beta 1----4galactosyltransferase (GalT-2) was investigated in rat liver. For this purpose, purified Golgi apparatus, endoplasmic reticulum, and plasma membrane fractions were prepared from the liver and used as the enzyme source for detecting GalT-2. A pure Golgi apparatus, highly enriched in many glycosyltransferases, was the only fraction where GalT-2 was measurable. The reaction product formation rate under appropriate assay conditions, which requires high detergent concentration and Mn2+, was low but comparable with that of other glycosyltransferases. The product formation was stimulated by exogenously added acceptor GlcCer, donor UDP-Gal, and Golgi protein. The reaction product was a single spot that was identified by chromatographic behavior, sensitivity to beta-galactosidase, and permethylation studies as Gal beta 1----4Glc beta 1----1'Cer (lactosylceramide). A metabolic experiment, performed by determining the glycosphingolipids which became radioactive in the above subcellular fractions prepared from the liver of animals treated with glucose-labeled glucosylceramide, further indicated that the in vivo glycosylation of glucosylceramide takes place in the Golgi apparatus.     

The Na+/H+ Exchanger NHE6 in the Endosomal Recycling System Is Involved in the Development of Apical Bile Canalicular Surface Domains in HepG2 Cells

Polarized epithelial cells develop and maintain distinct apical and basolateral surface domains despite a continuous flux of membranes between these domains. The Na⁺/H⁺exchanger NHE6 localizes to endosomes but its function is unknown. Here, we demonstrate that polarized hepatoma HepG2 cells express an NHE6.1 variant that localizes to recycling endosomes and colocalizes with transcytosing bulk membrane lipids. NHE6.1 knockdown or overexpression decreases or increases recycling endosome pH, respectively, and inhibits the maintenance of apical, bile canalicular plasma membranes and, concomitantly, apical lumens. NHE6.1 knockdown or overexpression has little effect on the de novo biogenesis of apical surface domains. NHE6.1 knockdown does not inhibit basolateral-to-apical transcytosis of bulk membrane lipids, but it does promote their progressive loss from the apical surface, leaving cells unable to efficiently retain bulk membrane and bile canalicular proteins at the apical surface. The data suggest that a limited range of endosome pH mediated by NHE6.1 is important for securing the polarized distribution of membrane lipids at the apical surface and maintenance of apical bile canaliculi in HepG2 cells and hence cell polarity. This study underscores the emerging role of the endosomal recycling system in apical surface development and identifies NHE6 as a novel regulatory protein in this process.     

Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface.

We show that a protein with a glycosylphosphatidyl inositol (GPI) anchor can be recovered from lysates of epithelial cells in a low density, detergent-insoluble form. Under these conditions, the protein is associated with detergent-resistant sheets and vesicles that contain other GPI-anchored proteins and are enriched in glycosphingolipids, but do not contain a basolateral marker protein. The protein is recovered in this complex only after it has been transported to the Golgi complex, suggesting that protein-sphingolipid microdomains form in the Golgi apparatus and plasma membrane and supporting the model proposed by Simons and colleagues for sorting of certain membrane proteins to the apical surface after intracellular association with glycosphingolipids.     

Glucosylceramide is synthesized at the cytosolic surface of various Golgi subfractions

In our attempt to assess the topology of glucosylceramide biosynthesis, we have employed a truncated ceramide analogue that permeates cell membranes and is converted into water soluble sphingolipid analogues both in living and in fractionated cells. Truncated sphingomyelin is synthesized in the lumen of the Golgi, whereas glucosylceramide is synthesized at the cytosolic surface of the Golgi as shown by (a) the insensitivity of truncated sphingomyelin synthesis and the sensitivity of truncated glucosylceramide synthesis in intact Golgi membranes from rabbit liver to treatment with protease or the chemical reagent DIDS; and (b) sensitivity of truncated sphingomyelin export and insensitivity of truncated glucosylceramide export to decreased temperature and the presence of GTP-gamma-S in semiintact CHO cells. Moreover, subfractionation of rat liver Golgi demonstrated that the sphingomyelin synthase activity was restricted to fractions containing marker enzymes for the proximal Golgi, whereas the capacity to synthesize truncated glucosylceramide was also found in fractions containing distal Golgi markers. A similar distribution of glucosylceramide synthesizing activity was observed in the Golgi of the human liver derived HepG2 cells. The cytosolic orientation of the reaction in HepG2 cells was confirmed by complete extractability of newly formed NBD-glucosylceramide from isolated Golgi membranes or semiintact cells by serum albumin, whereas NBD-sphingomyelin remained protected against such extraction.     

Targeting and fusion proteins during mammalian spermiogenesis.

Regulated exocytosis is controlled by internal and external signals. The molecular machinery controlling the sorting from the newly synthesized vesicles from the Golgi apparatus to the plasma membrane play a key role in the regulation of both the number and spatial location of the vesicles. In this context the mammalian acrosome is a unique vesicle since it is the only secretory vesicle attached to the nucleus. In this work we have studied the membrane trafficking between the Golgi apparatus and the acrosome during mammalian spermiogenesis. During bovine spermiogenesis, Golgi antigens (mannosidase II) were detected in the acrosome until the late cap-phase spermatids, but are not found in testicular spermatozoa (maturation-phase spermatids). This suggests that Golgiacrosome flow may be relatively unselective, with Golgi residents retrieved before spermination is complete. Surprisingly, rab7, a protein involved in lysosome/endosome trafficking was also found associated with the acrosomal vesicle during mouse spermiogenesis. Our results suggest that the acrosome biogenesis is associated with membrane flow from both the Golgi apparatus and the endosome/lysosome system in mammalian spermatids.