Prosaposin: a protein with differential sorting and multiple functions.

In eukaryotes the delivery of newly synthesized proteins to their final destination is dependent on a series of functionally distinct compartments, including the endoplasmic reticulum and the Golgi apparatus, which plays a role in post-translational modification, sorting and distribution of proteins. Most cargo is sorted within, and exits from, the trans-Golgi network (TGN). Proteins delivered to lysosomes include hydrolytic enzymes and nonenzymic activator proteins. They are directed away from the cell surface by their binding to mannose-6-phosphate receptors (MPR). However, in I-cell disease, in which the MPR pathway is disrupted, the nonenzymic sphingolipid activator protein, prosaposin, continue to traffic to lysosomes. This observation led to discovery of a new lysosomal sorting receptor, sortilin. The targeting prosaposin to the lysosomes results from the interaction of its C-terminus with sortilin. Deletion of the C-terminus did not interfere with its secretion, but abolished its transport to the lysosomes. Mutational analysis revealed that the first half of the prosaposin C-terminus contains a motif required for its binding to sortilin and its transport to the lysosomes. Prosaposin can be also secreted to the extracellular space as oligomers. Extracellular prosaposin showed to exert a variety of responses in nervous tissues including the activation of G protein-coupled receptors and ERK phosphorylation. Lastly, prosaposin has been found to be expressed in other fluids of the body such as pancreatic juice, bile, cerebrospinal fluid, milk and seminal fluid, indicating that prosaposin is not only a house keeping lysosomal protein but an essential factor in the development and maintenance of the nervous systems and other systems of the body.

The sorting and trafficking of lysosomal proteins.

For a long time lysosomes were considered terminal organelles involved in the degradation of different substrates. However, this view is rapidly changing by evidence demonstrating that these organelles and their content display specialized functions in addition to the degradation of substances. Many lysosomal proteins have been implicated in specialized cellular functions and disorders such as antigen processing, targeting of surfactant proteins, and most lysosomal storage disorders. To date, about fifty lysosomal hydrolases have been identified, and the majority of them are targeted to the lysosomes via the mannose-6-phosphate receptor (M6P-Rc). However, recent studies on the intracellular trafficking of the non-enzymic lysosomal proteins prosaposin and GM2 activator (GM2AP) demonstrated that they use an alternative receptor termed "sortilin". Existing evidence suggests that some hydrolases traffic to the lysosomes in a mannose 6-phophate-indepentend manner. The possibility that sortilin is implicated in the targeting of some soluble hydrolases, as well as the consequences of this process, is addressed in the present review.

In-vivo effects of Glu298Asp endothelial nitric oxide synthase polymorphism.

Endothelial nitric oxide synthase catalyses the formation of the vasodilator nitric oxide, a major regulator of vascular tone. The Asp298 polymorphism of the nitric oxide synthase gene is associated with altered function and expression of the enzyme in vitro and myocardial infarction and coronary artery spasm in vivo. We examined the effect of the Glu298Asp polymorphism on: (1) local vascular responses to phenylephrine, acetylcholine, glyceryl trinitrate and prostaglandin E1 in the dorsal hand vein; (2) changes in forearm blood flow during mental stress, a measure of nitric oxide-mediated effect on resistance vessels; (3) excretion of urinary nitrite/nitrate as a measure of total body nitric oxide production; and (4) F2-isoprostane metabolite, a measure of oxidative stress, in healthy Glu298 (n = 12) and Asp298 (n = 13) homozygotes. There were no significant differences in acetylcholine dose responses (P = 0.29) in Glu298 and Asp298 homozygotes. Responses to glyceryl trinitrate, prostaglandin E1 and the alpha-adrenergic agonist phenylephrine did not differ by genotype. Forearm blood flow was similar at rest and increased significantly (from 7.5 ml/min/100 ml to 12.2 ml/min/100 ml; P = 0.003), but similarly (P = 0.2), during mental stress in both genotypes. Asp298 homozygotes excreted significantly less nitrate/nitrite than Glu298 homozygotes (nitrate + nitrite/creatinine ratio 0.05 +/- 0.01 vs. 0.09 +/- 0.01, respectively; P < 0.005). Urinary F2-isoprostane metabolite excretion did not differ (Glu298, 2.04 +/- 0.25 ng/mg creatinine; Asp298, 1.85 +/- 0.37 ng/mg creatinine; P = 0.7). We conclude that in healthy volunteers the Glu298Asp polymorphism affects endogenous nitric oxide production without affecting nitric oxide-mediated vascular responses. This polymorphism may only have clinical significance in the presence of endothelial dysfunction.

Improved assay for the quantification of the major urinary metabolite of the isoprostane 15-F(2t)-Isoprostane (8-iso-PGF(2alpha)) by a stable isotope dilution mass spectrometric assay.

BACKGROUND: The F(2)-isoprostanes (IsoPs) are a series of novel prostaglandin (PG)-like compounds generated from the free radical catalyzed peroxidation of arachidonic acid. One IsoP, 15-F(2t)-IsoP (8-iso-PGF(2alpha)), has been shown to be formed in abundance in vivo and to exert potent biological activity. METHODS: As a means to assess the endogenous production of this compound, we previously developed a method to quantify the major urinary metabolite of 15-F(2t)-IsoP, 2,3-dinor-5,6-dihydro-15-F(2t)-IsoP (2,3-dinor-5,6-dihydro-8-iso-PGF(2alpha), 15-F(2t)-IsoP-M ), by gas chromotography (GC)/negative ion chemical ionization mass spectrometry (MS) employing stable isotope dilution methodology. While useful, we found that the assay occasionally suffered from the presence of impurities that co-elute on GC with 15-F(2t)-IsoP-M, making the measurement of this compound difficult. We now report a modified assay for the quantification of 15-F(2t)-IsoP-M employing GC/MS that alleviates this problem. RESULTS: Precision of the assay is +/-7% and the accuracy is 96%. The lower limit of sensitivity is approximately 8 pg. Normal concentrations of this metabolite in urine were found to be 0.46+/-0.09 ng/mg creatinine (mean+/-1 S.D.) Urinary excretion of 15-F(2t)-IsoP-M is markedly altered in situations associated with increased or decreased oxidant stress in vivo. CONCLUSIONS: This assay provided a sensitive and accurate method to assess endogenous IsoP generation and can be used to further explore the role of oxidant injury in human disease.

Intracellular distribution of lysosomal sialidase is controlled by the internalization signal in its cytoplasmic tail.

Sialidase (neuraminidase), encoded by the neu-1 gene in the major histocompatibility complex locus catalyzes the intralysosomal degradation of sialylated glycoconjugates. Inherited deficiency of sialidase results in sialidosis or galactosialidosis, both severe metabolic disorders associated with lysosomal storage of oligosaccharides and glycopeptides. Sialidase also plays an important role in cellular signaling and is specifically required for the production of cytokine interleukin-4 by activated T lymphocytes. In these cells, neu-1-encoded sialidase activity is increased on the cell surface, suggesting that a specific mechanism regulates sorting of this enzyme to the plasma membrane. We investigated that mechanism by first showing that sialidase contains the internalization signal found in lysosomal membrane proteins targeted to endosomes via clathrin-coated pits. The signal consists of a C-terminal tetrapeptide (412)YGTL(415), with Tyr(412) and Leu(415) essential for endocytosis of the enzyme. We further demonstrated that redistribution of sialidase from lysosomes to the cell surface of activated lymphocytes is accompanied by increased reactivity of the enzyme with anti-phosphotyrosine antibodies. We speculate that phosphorylation of Tyr(412) results in inhibition of sialidase internalization in activated lymphocytes.

Delay in sexual maturity of the follicle-stimulating hormone receptor knockout male mouse.

In the highly organized and complex process of mammalian spermatogenesis, the development of an undifferentiated diploid germ cell into a fully differentiated and mature spermatozoon is orchestrated in a time frame unique for each species including man. If the various hormonal signals including environmental cues that play a critical part in initiating these events are not properly executed, various deficiencies including delay in sexual maturity or puberty are likely. In this study we have followed testicular development and spermatogenesis in the FSH receptor knockout (FORKO) mice from Day 7 onward by using histology and quantitative DNA flow cytometry. The drastic reduction in testicular weight and shrinkage of seminiferous tubules that occurred at this early age persisted into the adult stage in the FORKOs, suggesting inhibition of the initial developmental processes. The round spermatids that were clearly abundant on Day 21 in the wild-type and heterozygous males were few and present only in some tubules of the FORKOs. There were no elongated spermatids in FORKO males on Day 35. The sperm produced by Day 49 FORKOs were already aberrant, a feature that persisted into adulthood in these animals. As all these changes occurred in a background of normal circulating testosterone levels, we may conclude that the delay in testicular development is a consequence of the loss of FSH-receptor signaling. The delay in sexual maturity of FORKOs was accompanied by reduction in fertility as evidenced by mating studies. Based on these data we suggest that the FORKO mouse might be a useful experimental model to define the molecular mechanisms that underlie the delay in puberty.

Biogenesis of lysosomes in marshall cells and in cells of the male reproductive system.

UNLABELLED: The mechanism of plasma membrane trafficking and degradation is still poorly understood. This investigation deals with the biogenesis of lysosomes during endocytic flow in Marshall cells and in various cell types of the male reproductive system. Marshall cells were exposed to ammonium chloride (NH4Cl) and leupeptin after labeling with cationic ferritin. In some experiments, the treated cells were immunogold labeled with anti-prosaposin antibody. NH4Cl and leupeptin are lysosomotropic agents that affect the endosomal-lysosomal progression. Testes, efferent ducts and epididymis from mouse mutants with defects affecting plasma membrane degradation were also used to analyze this process. NH4Cl produced a retention of cationic ferritin in endosomes and hindered the endosomal/lysosomal progression. Leupeptin did not affect this process. NH4Cl decreased the labeling of prosaposin in endosomes and lysosomes, while leupeptin increased the labeling of prosaposin in lysosomes. The number of lysosomes per cytoplasmic area was higher in treated cells than in controls. These findings suggest that leupeptin affected lysosomes whereas NH4Cl affected both endosomes and lysosomes. The endosomal and lysosomal accumulation of prosaposin induced by the treatment with NH4Cl and leupeptin indicated that the site of entry of prosaposinwas both the lysosome and endosome. Electron microscopy (EM) of tissues from mouse mutants with defects affecting plasma membrane degradation substantiated these observations. The EM analysis revealed a selective accumulation of multivesicular bodies (MVBs) and the disappearance of lysosomes, in testicular fibroblasts, nonciliated cells of the efferent ducts and principal cells of the epididymis, suggesting that MVBs are precursors of lysosomes. IN CONCLUSION: (1) endosomes and MVBs are a required steps for degradation of membranes; (2) endosomes and MVBs are precursors of lysosomes; and (3) endosomes, MVBs, and lysosomes appear to be transient organelles.

Mutations in sialidosis impair sialidase binding to the lysosomal multienzyme complex.

Sialidosis is an autosomal recessive disease caused by the genetic deficiency of lysosomal sialidase, which catalyzes the catabolism of sialoglycoconjugates. The disease is associated with progressive impaired vision, macular cherry-red spots, and myoclonus (sialidosis type I) or with skeletal dysplasia, Hurler-like phenotype, dysostosis multiplex, mental retardation, and hepatosplenomegaly (sialidosis type II). We analyzed the effect of the missense mutations G68V, S182G, G227R, F260Y, L270F, A298V, G328S, and L363P, which are identified in the sialidosis type I and sialidosis type II patients, on the activity, stability, and intracellular distribution of sialidase. We found that three mutations, F260Y, L270F, and A298V, which are clustered in the same region on the surface of the sialidase molecule, dramatically reduced the enzyme activity and caused a rapid intralysosomal degradation of the expressed protein. We suggested that this region might be involved in sialidase binding with lysosomal cathepsin A and/or beta-galactosidase in the multienzyme lysosomal complex required for the expression of sialidase activity. Transgenic expression of mutants followed by density gradient centrifugation of cellular extracts confirmed this hypothesis and showed that sialidase deficiency in some sialidosis patients results from disruption of the lysosomal multienzyme complex.

Elevated levels of the polyadenylation factor CstF 64 enhance formation of the 1kB Testis brain RNA-binding protein (TB-RBP) mRNA in male germ cells.

The single copy mouse Testis Brain RNA-Binding Protein (TB-RBP) gene encodes three mRNAs of 3.0, 1.7, and 1.0 kb which only differ in their 3' UTRs. The 1 kb TB-RBP mRNA predominates in testis, while somatic cells preferentially express the 3.0 kb TB-RBP mRNA. Here we show that the 1 kb mRNA is translated several-fold more efficiently than the 3 kb TB-RBP in rabbit reticulocyte lysates and cells with elevated levels of the 1 kB TB-RBP mRNA express high levels of TB-RBP. To determine if the cleavage stimulatory factor CstF 64 can modulate the alternative splicing of the TB-RBP pre-mRNA and therefore TB-RBP expression, CstF 64 levels and binding to alternative polyadenylation sites were examined. CstF 64 is abundant in the testis and preferentially binds to a distal site in the TB-RBP pre-mRNA that produces the 3 kb TB-RBP. Moreover, upregulation or overexpression of CstF 64 increases the poly(A) site selection for the 1 kb TB-RBP mRNA. We propose that the level of the polyadenylation factor CstF 64 modulates the level of TB-RBP synthesis in male germ cells by an alternative processing of the TB-RBP pre-mRNA.

Targeted disruption of the mouse prosaposin gene affects the development of the prostate gland and other male reproductive organs.

The prosaposin gene encodes a 65-70 kilodalton (kd) protein, which is secreted or targeted to lysosomes. In lysosomes, prosaposin is the precursor of 4 activator proteins, designated saposins A, B, C, and D, which promote by acidic hydrolases, the degradation of glycosphingolipids with short oligosaccharide chains. Mutations of the prosaposin gene have been linked to several lysosomal storage disorders. An animal model was recently developed by creating a null allele in embryonic stem cells through gene targeting in order to investigate the phenotypic diversity of prosaposin mutations, the involvement of this protein in lysosomal storage diseases, and to develop potential therapeutic approaches. Mutant homozygous mice die at 35-40 days of age and neurological disorders contribute to their early death. Secreted prosaposin is present in milk and in cerebrospinal and seminal fluids. In the nervous system, prosaposin exhibits a trophic activity. Examination of reproduc-tive organs in homozygous mutant males shows several abnormalities such as a decrease in testis size with reduced spermiogenesis, and an involution of the prostate, seminal vesicle, and epididymis, although levels of testosterone in blood remain normal. In the prostate of homozygous mutants, only basal cells appear to be present, whereas secretory cells are absent. The epithelia in efferent ducts is formed by ciliated cells, whereas heterozygotes exhibit a majority of nonciliated cells. Our data indicate that prosaposin is involved in the development and maintenance of male reproductive organs. In prostatic epithelium, targeted disruption of the prosaposin gene appears to inactivate the mitogen-activated protein kinase pathway and to interfere with differentiation of secretory cells.

Congeners of N(alpha)-acetyl-L-cysteine but not aminoguanidine act as neuroprotectants from the lipid peroxidation product 4-hydroxy-2-nonenal.

Increased generation of neurotoxic lipid peroxidation products is proposed to contribute to the pathogenesis of Alzheimer's disease (AD). Current antioxidant therapies are directed at limiting propagation of brain lipid peroxidation. Another approach would be to scavenge the reactive aldehyde products of lipid peroxidation. N(alpha)-acetyl-L-cysteine (NAC) and aminoguanidine (AG) react rapidly and irreversibly with 4-hydroxy-2-nonenal (HNE) in vitro, and both have been proposed as potential scavengers of HNE in biological systems. We have compared NAC, AG, and a series of congeners as scavengers of HNE and as neuroprotectants from HNE. Our results showed that while both NAC and AG had comparable chemical reactivity with HNE, only NAC and its congeners were able to block HNE-protein adduct formation in vitro and in neuronal cultures. Moreover, NAC and its congeners, but not AG, effectively protected brain mitochondrial respiration and neuronal microtubule structure from the toxic effects of HNE. We conclude that NAC and its congeners, but not AG, may act as neuroprotectants from HNE.

Divergent N-terminal sequences target an inducible testis deubiquitinating enzyme to distinct subcellular structures.

Ubiquitin-specific processing proteases (UBPs) presently form the largest enzyme family in the ubiquitin system, characterized by a core region containing conserved motifs surrounded by divergent sequences, most commonly at the N-terminal end. The functions of these divergent sequences remain unclear. We identified two isoforms of a novel testis-specific UBP, UBP-t1 and UBP-t2, which contain identical core regions but distinct N termini, thereby permitting dissection of the functions of these two regions. Both isoforms were germ cell specific and developmentally regulated. Immunocytochemistry revealed that UBP-t1 was induced in step 16 to 19 spermatids while UBP-t2 was expressed in step 18 to 19 spermatids. Immunoelectron microscopy showed that UBP-t1 was found in the nucleus while UBP-t2 was extranuclear and was found in residual bodies. For the first time, we show that the differential subcellular localization was due to the distinct N-terminal sequences. When transfected into COS-7 cells, the core region was expressed throughout the cell but the UBP-t1 and UBP-t2 isoforms were concentrated in the nucleus and the perinuclear region, respectively. Fusions of each N-terminal end with green fluorescent protein yielded the same subcellular localization as the native proteins, indicating that the N-terminal ends were sufficient for determining differential localization. Interestingly, UBP-t2 colocalized with anti-gamma-tubulin immunoreactivity, indicating that like several other components of the ubiquitin system, a deubiquitinating enzyme is associated with the centrosome. Regulated expression and alternative N termini can confer specificity of UBP function by restricting its temporal and spatial loci of action.

Role of prosaposin in the male reproductive system: effect of prosaposin inactivation on the testis, epididymis, prostate, and seminal vesicles.

SGP-1/prosaposin can be secreted or targeted to the lysosomes where it is processed into smaller saposins (A, B, C, and D) required for the hydrolysis of glycosphingolipids. The deficiency of saposins B and C results in variant forms of metachromatic leukodystrophy and Gaucher's disease, respectively, which are characterized by lysosomal storage of undegraded glycosphingolipids. In the nervous system, prosaposin presents trophic activity. A mouse model was recently developed by creating a null allele in embryonic stem cells through gene targeting to investigate the phenotypic diversity of prosaposin mutations and the involvement of this protein in lysosomal storage diseases, and for the development of therapeutic approaches. Mice homozygous mutants die at the age of 35-40 days and neurological disorders contribute to the early demise of the mutant mice. The male reproductive organs in homozygous mutants show several abnormalities, such as a decrease in testis size with reduced spermiogenesis and an involution of the prostate, seminal vesicles, and epididymis. In these animals, the blood levels of testosterone remain normal. In the prostate of homozygous mutants, only the basal epithelial cells appear to be present, while the secretory cells are absent. These findings suggest that prosaposin may be involved in the development and maintenance of the male reproductive organs, as well as, in cellular differentiation.

Identification of a novel sequence involved in lysosomal sorting of the sphingolipid activator protein prosaposin.

Prosaposin is synthesized as a 53-kDa protein, post-translationally modified to a 65-kDa form and further glycosylated to a 70-kDa secretory product. The 65-kDa protein is associated to Golgi membranes and is targeted to lysosomes, where four smaller nonenzymatic saposins implicated in the hydrolysis of sphingolipids are generated by its partial proteolysis. The targeting of the 65-kDa protein to lysosomes is not mediated by the mannose 6-phosphate receptor. The Golgi apparatus appears to accomplish the molecular sorting of the 65-kDa prosaposin by decoding a signal from its amino acid backbone. This investigation deals with the characterization of the sequence involved in this process by deleting the saposin functional domains A, B, C, and D and the highly conserved N and C termini of prosaposin. The truncated cDNAs were subcloned into expression vectors and transfected to COS-7 cells. The destination of the mutated proteins was assessed by immunocytochemistry. Deletion of the C terminus did not interfere with the secretion of prosaposin but abolished its transport to lysosomes. Deletion of saposins and the N-terminal domain did not affect the lysosomal or secretory routing of prosaposin. A chimeric construct of albumin and the C terminus of prosaposin was not directed to lysosomes. However, albumin connected to the C terminus and one or more functional domains of prosaposin reached lysosomes, indicating that the C terminus and at least one saposin domain are required for this process. In summary, we are reporting a novel sequence involved in the targeting of prosaposin to lysosomes.

Qualitative and quantitative decline in spermatogenesis of the follicle-stimulating hormone receptor knockout (FORKO) mouse.

Sertoli cells express functional receptors for FSH, one of the two pituitary hormones that regulate spermatogenesis in mammals. We recently produced genetic mutant (FORKO) mice that lack FSH receptor, in order to examine the effects on testicular function and fertility. Mutant males exhibited weight loss of testis, epididymis, and seminal vesicle as well as low levels of testosterone. Except for reduced seminiferous tubular diameter, no gross changes were apparent upon histological examination. Analysis of testicular germ cells by flow cytometry revealed a significant increase in the percentage of 2C cells (spermatogonia and non-germ cells) and a significant decrease in the percentage of HC cells (elongated spermatids) of FORKO males. The absolute number of homogenization-resistant elongated spermatids was also significantly reduced in the mutant males. A 2-fold increase in c-kit-positive 2C cells was recorded in the mutant males. Elongated spermatids of FORKO males showed a dramatic increase in propidium iodide binding suggesting reduced nuclear compaction. The increase in size of the sperm head in mutants, as well as susceptibility to dithiothreitol-induced decondensation, suggests the inadequate condensation of sperm chromatin. Sperm chromatin structure assay, a technique that reflects DNA stability, revealed that sperm from FORKO males are susceptible to acid denaturation, indicating the poor quality of sperm. These data allow us to conclude that genetic disruption of FSH receptor signaling in the rodent induces major changes that might contribute to reduced fertility.

Circulating and luminal testicular factors affect LRP-2 and Apo J expression in the epididymis following efferent duct ligation.

Apolipoprotein J (clusterin or sulfated glycoprotein-2) has been shown to be secreted by the epididymal principal cells, whereupon it binds to sperm in the lumen. Apolipoprotein J also is endocytosed by principal cells along the epididymis. Recently, it has been demonstrated that low-density lipoprotein receptor-related protein-2 (LRP-2) mediates the endocytosis of Apo J and is present in the epididymis. The purpose of the present study was to determine the factors regulating the synthesis of these 2 proteins in various experimentally treated animals. The epididymides of adult rats were fixed with Bouin's fluid and examined with anti-Apo J and anti-LRP-2 antibodies by a light microscope immunocytochemical method. In normal adult animals, expression of Apo J was evident in principal cells of all epididymal regions except the proximal initial segment. Diffuse cytoplasmic staining indicated Apo J secretion. Reactive apical vesicles, presumably endosomal in nature, suggested endocytosis of Apo J. Lipoprotein receptor-related protein-2 expression was solely apical in nature and was seen as an intense apical band in principal cells of all regions except the proximal and distal initial segment and distal caput regions of the epididymis. Hypophysectomy, up to 28 days after the procedure, did not affect expression of Apo J or LRP-2 in principal cells along the entire epididymis. Orchidectomy, with or without testosterone replacement at all time intervals examined, also did not affect LRP-2 expression along the entire epididymis. This also was noted for Apo J expression in all regions except the proximal initial segment. Thus, expression of these 2 proteins does not appear to be regulated by testicular or pituitary factors. In contrast, bilateral as well as unilateral (intact and ligated sides) efferent duct ligation resulted in dramatic differences in LRP-2 and Apo J expression in principal cells in the various epididymal regions. In the case of LRP-2, a complete absence of reaction was noted in principal cells along the entire epididymis. As for Apo J, expression in the distal initial segment, intermediate zone, and caput region remained unchanged compared with that in normal adult animals, whereas in the corpus and cauda epididymides, results of cytoplasmic staining were negligible. These results suggest that under conditions of efferent duct ligation, a circulating factor emanates from the testis to inhibit expression of LRP-2 and Apo J in these epididymal regions. Furthermore, because Apo J was affected in a region-specific manner, unlike the case for LRP-2, different factors appear to be involved for each protein. These factors may be produced to inhibit proteins from being synthesized by the epididymis in the absence of luminal testicular input and may exist in cases of congenital and pathologic epididymal tubule blockages as well as after vasectomy. In the case of immunostaining for Apo J in the proximal initial segment only, normally unreactive principal cells in control adult animals became intensely reactive after orchidectomy as well as bilateral and unilateral (ligated side only) ligation. As this was not the case for hypophysectomized animals and the intact side of unilateral efferent duct-ligated animals, it is suggested that a testicular factor entering via the lumen of the efferent ducts serves to inhibit Apo J expression in this area. The present data also reveal that after efferent duct ligation, there are circulating factors that inhibit Apo J expression in a region-specific manner (corpus and cauda) and that inhibit LRP-2 expression along the entire epididymis and that these are derived from the testis. Furthermore, the data reveal that a testicular luminal factor appears to inhibit Apo J expression in the proximal initial segment of normal adult animals. Key words: Principal cells, orchidectomy, glycoprotein 330, clusterin, sulfated glycoprotein-2.

Biogenesis of lysosomes by endocytic flow of plasma membrane

The objective of this review is to examine the biogenesis of lysosomes during the endocytic flow of plasma membrane. Two models have been proposed to explain the formation of lysosomes by this process: the "maturational" and the "stationary" models. According to the former, pinocytotic vesicles fuse among themselves to yield endosomes, which in turn, transform first into multivesicular bodies (MVB) and then into mature lysosomes. Therefore, endosomes and lysosomes would be transient organelles. On the other hand, the "stationary" model proposes that the endocytic pathway is formed by functionally and physically distinct compartments. Cultured cells exposed to ammonium chloride (NH4Cl) and leupeptin after a pulse of cationic ferritin were recently used to freeze endosomes and lysosomes. NH4Cl produced a retention of cationic ferritin in endosomes, indicating that this agent interfered with the endosomal/lysosomal progression. Leupeptin did not affect this process. The number of lysosomes increased in cells treated with both lysosomotropic agents. Thus, NH4Cl affected the endosomal and lysosomal compartments, whereas leupeptin had a preferential effect on lysosomes. Mice mutants with defects of plasma membrane degradation, including a Tay-Sachs model, a Sandhoff disease model, as well as, mice with the inactivated prosaposin gene were used to analyze the biogenesis of lysosomes. Thin sections of mutant cells were examined under the electron microscope, and the analysis revealed a selective accumulation of MVBs and the disappearance of lysosomes, suggesting that the formation of MVBs is a required step in lysosomal maturation and that the intravesicular content of MVBs is necessary for the digestion of plasma membrane components. Taken together, these data indicate that endosomes and MVBs are preceding steps in lysosomal biogenesis and that endosomes, MVBs, and lysosomes are transient organelles

Biogenesis of lysosomes by endocytic flow of plasma membrane

The objective of this review is to examine the biogenesis of lysosomes during the endocytic flow of plasma membrane. Two models have been proposed to explain the formation of lysosomes by this process: the "maturational" and the "stationary" models. According to the former, pinocytotic vesicles fuse among themselves to yield endosomes, which in turn, transform first into multivesicular bodies (MVB) and then into mature lysosomes. Therefore, endosomes and lysosomes would be transient organelles. On the other hand, the "stationary" model proposes that the endocytic pathway is formed by functionally and physically distinct compartments. Cultured cells exposed to ammonium chloride (NH4Cl) and leupeptin after a pulse of cationic ferritin were recently used to freeze endosomes and lysosomes. NH4Cl produced a retention of cationic ferritin in endosomes, indicating that this agent interfered with the endosomal/lysosomal progression. Leupeptin did not affect this process. The number of lysosomes increased in cells treated with both lysosomotropic agents. Thus, NH4Cl affected the endosomal and lysosomal compartments, whereas leupeptin had a preferential effect on lysosomes. Mice mutants with defects of plasma membrane degradation, including a Tay-Sachs model, a Sandhoff disease model, as well as, mice with the inactivated prosaposin gene were used to analyze the biogenesis of lysosomes. Thin sections of mutant cells were examined under the electron microscope, and the analysis revealed a selective accumulation of MVBs and the disappearance of lysosomes, suggesting that the formation of MVBs is a required step in lysosomal maturation and that the intravesicular content of MVBs is necessary for the digestion of plasma membrane components. Taken together, these data indicate that endosomes and MVBs are preceding steps in lysosomal biogenesis and that endosomes, MVBs, and lysosomes are transient organelles

Role of sphingolipids in the transport of prosaposin to the lysosomes.

Prosaposin is the precursor of four lysosomal saposins that promote the degradation of glycosphingolipids (GSLs) by acidic hydrolases. GSLs contain a hydrophobic ceramide moiety, which acts as a membrane anchor, and a hydrophilic oligosaccharide chain that faces the lumen of the Golgi apparatus and extracellular spaces. By using fumonisin B1, PDMP and D609, we tested the hypothesis that sphingolipids mediate the transport of prosaposin to the lysosomes. Fumonisin B1 interferes with the synthesis of ceramide, PDMP blocks the formation of glucosylceramide and D609 blocks the formation of sphingomyelin. Fumonisin B1 produced a 59;-85% decrease in the density of gold particles in the lysosomes of CHO and NRK cells immunolabeled with anti-prosaposin antibody, and a 55% reduction in the lysosomes of CHO cells stably transfected with an expression vector containing a human prosaposin cDNA. To examine whether the mannose 6-phosphate receptor pathway was affected by this treatment, NRK and CHO cells treated or not with fumonisin B1 were labeled with anti-cathepsin A antibody. The results showed no significant differences in labeling of the lysosomes, suggesting that the effect of fumonisin B1 was specific. When fumonisin B1 and D609 were added to the media of transfected CHO cells, a decrease in immunofluorescence with anti-prosaposin antibody was observed by confocal microscopy. PDMP did not cause any reduction in immunoreactivity, indicating that sphingolmyelin appears to be involved in this process. In conclusion, our data support the hypothesis that sphingolipids, possibly sphingomyelin, are involved in the transport of prosaposin to the lysosomes.

Protein-protein interactions between the testis brain RNA-binding protein and the transitional endoplasmic reticulum ATPase, a cytoskeletal gamma actin and Trax in male germ cells and the brain.

Numerous functions have been proposed for the testis brain RNA-binding protein (TB-RBP) and its human homologue, Translin, ranging from mRNA transport and translational regulation to DNA rearrangement and repair. To gain insight into the likely functions of this 26 kDa protein, immunoprecipitation was used to identify proteins that interact with TB-RBP in mouse cytosolic extracts. Three proteins, the transitional endoplasmic reticulum ATPase, a cytoskeletal gamma actin, and Trax, were specifically immunoprecipitated with an affinity-purified antibody to recombinant mouse TB-RBP. In vitro binding assays with recombinant proteins and EM immunocytochemistry confirm that TB-RBP interacts with the TER ATPase in vitro and in vivo. Confocal microscopy has demonstrated that TB-RBP colocalizes with actin in the cytoplasm of male germ cells. The immunoprecipitation of Trax with TB-RBP confirms a published report demonstrating protein interactions between the two proteins in a yeast two-hybrid assay. These data support the hypothesis that TB-RBP serves as a link in attaching specific mRNAs to cytoskeletal structures and suggests an involvement for the ubiquitously expressed TER ATPase in intracellular and/or intercellular mRNA transport.