Dark side of the epididymis: tails of sperm maturation.

BACKGROUND: The Hermes body (HB) previously called the cytoplasmic droplet is a focal distension of the flagellar cytoplasm of epididymal spermatozoa consisting mainly of isolated flattened Golgi cisternae. OBJECTIVE: To define a functional role for the HB of epididymal spermatozoa. METHODS: Isolated fractions of HBs of epididymal spermatozoa were prepared and by quantitative tandem mass spectrometry revealed 1511 proteins. RESULTS: The glucose transporter GLUT-3 was the most abundant protein followed by hexokinase 1, which along with the presence of all glycolytic enzymes suggested a role for the HB in glycolysis. Several TMED/p24 Golgi trafficking proteins were abundant with TMED7/p27 and TMED2/p24 defining the identity of the flattened cisternae within the HB as Golgi, along with the known Golgi proteins, GBF1, GOLPH3, Man2α1, and ManIIX. The Golgi trafficking protein TMED7/p27 via small 50-nm vesicles emanating from the Golgi cisternae was proposed to transport GLUT-3 to the plasma membrane for ATP production related to sperm motility. The internal membranes revealed abundant proteins not only of Golgi cisternae, but also of endoplasmic reticulum and endosomes. COPI and COPII coats, clathrin, SNAREs, annexins, atlastins, and GTPases were identified for vesicular trafficking and membrane fusion, in addition to ribosomes, stress proteins for protection, proteasome proteins involved in degradation, and cytoskeletal elements for migration of the HB along the flagellum. The biogenesis of the HB occurring at step 19 spermatids of the testis just prior to their release was uncovered as a key step in germ cell differentiation, where several proteins were expressed, some for the first time. CONCLUSION: As epididymal spermatozoa undergo remodeling of their protein makeup through selective degradation of sperm proteins during epididymal transit, then remodeling as a consequence of new protein synthesis is not excluded by our observations.

Association of calnexin with mutant peripheral myelin protein-22 ex vivo: a basis for "gain-of-function" ER diseases.

Schwann cell-derived peripheral myelin protein-22 (PMP-22) when mutated or overexpressed causes heritable neuropathies with a previously unexplained "gain-of-function" endoplasmic reticulum (ER) retention phenotype. In wild-type sciatic nerves, PMP-22 associates in a specific, transient (t(1/2 ) approximately equal to 11 min), and oligosaccharide processing-dependent manner with the lectin chaperone calnexin (CNX), but not calreticulin nor BiP. In Trembler-J (Tr-J) sciatic nerves, prolonged association of mutant PMP-22 with CNX is found (t(1/2) > 60 min). In 293A cells overexpressing PMP-22(Tr-J), CNX and PMP-22 colocalize in large intracellular structures identified at the electron microscopy level as myelin-like figures with CNX localization in the structures dependent on PMP-22 glucosylation. Similar intracellular myelin-like figures were also present in Schwann cells of sciatic nerves from homozygous Trembler-J mice with no detectable activation of the stress response pathway as deduced from BiP and CHOP expression. Sequestration of CNX in intracellular myelin-like figures may be relevant to the autosomal dominant Charcot-Marie-Tooth-related neuropathies.

Localization of specific binding sites for 125I-TGF-beta1 to fenestrated endothelium in bone and anastomosing capillary networks in enamel organ suggests a role for TGF-beta1 in angiogenesis.

Previous studies have shown endothelial cells to be a major target for endocrine TGF-beta in several soft tissues in the normal growing rat. The potent effect of TGF-beta1 on bone formation prompted us to analyze in detail the localization of specific binding sites for endocrine TGF-beta in hard tissues. At 2.5 minutes after injection of 125I-TGF-beta1, specific binding, as demonstrated by quantitative radioautography, was localized to fenestrated endothelium participating in angiogenesis in the vascular invasion region of the growth plate in bone as well as to anatomizing capillary networks in the maturation zone of the enamel organ. At 15 minutes after injection, the bound ligand was internalized into endocytic vesicles of endothelial cells. In bone, quantitation revealed significant differences in receptor density between endothelia undergoing proliferation vs those in a state of elongation and anastomosis with neighboring endothelial cells. In the rat incisor, specific binding of 125I-TGF-beta1 to endothelium correlated with increased formation of anastomotic capillary networks. These studies identify differential specific binding sites of 125I-TGF-beta1 in angiogenically active endothelium, providing an important link between TGF-beta1, the endothelium, and hard tissue development.

The Structure of calnexin, an ER chaperone involved in quality control of protein folding.

The three-dimensional structure of the lumenal domain of the lectin-like chaperone calnexin determined to 2.9 A resolution reveals an extended 140 A arm inserted into a beta sandwich structure characteristic of legume lectins. The arm is composed of tandem repeats of two proline-rich sequence motifs which interact with one another in a head-to-tail fashion. Identification of the ligand binding site establishes calnexin as a monovalent lectin, providing insight into the mechanism by which the calnexin family of chaperones interacts with monoglucosylated glycoproteins.

Htm1p, a mannosidase-like protein, is involved in glycoprotein degradation in yeast.

Misfolded proteins are recognized in the endoplasmic reticulum (ER), transported back to the cytoplasm and degraded by the proteasome. Processing intermediates of N-linked oligosaccharides on incompletely folded glycoproteins have an important role in their folding/refolding, and also in their targeting to proteolytic degradation. In Saccharomyces cerevisiae, we have identified a gene coding for a non-essential protein that is homologous to mannosidase I (HTM1) and that is required for degradation of glycoproteins. Deletion of the HTM1 gene does not affect oligosaccharide trimming. However, deletion of HTM1 does reduce the rate of degradation of the mutant glycoproteins such as carboxypeptidase Y, ABC-transporter Pdr5-26p and oligosaccharyltransferase subunit Stt3-7p, but not of mutant Sec61-2p, a non-glycoprotein. Our results indicate that although Htm1p is not involved in processing of N-linked oligosaccharides, it is required for their proteolytic degradation. We propose that this mannosidase homolog is a lectin that recognizes Man8GlcNAc2 oligosaccharides that serve as signals in the degradation pathway.

Inhibition of endosomal insulin-like growth factor-I processing by cysteine proteinase inhibitors blocks receptor-mediated functions.

The receptor for the type 1 insulin-like growth factor (IGF-I) has been implicated in cellular transformation and the acquisition of an invasive/metastatic phenotype in various tumors. Following ligand binding, the IGF-I receptor is internalized, and the receptor.ligand complex dissociates as the ligand is degraded by endosomal proteinases. In the present study we show that the inhibition of endosomal IGF-I-degrading enzymes in human breast and murine lung carcinoma cells by the cysteine proteinase inhibitors, E-64 and CA074-methyl ester, profoundly altered receptor trafficking and signaling. In treated cells, intracellular ligand degradation was blocked, and although the receptor and two substrates, Shc and Insulin receptor substrate, were hyperphosphorylated on tyrosine, IGF-I-induced DNA synthesis, anchorage-independent growth, and matrix metalloproteinase synthesis were inhibited. The results suggest that ligand processing by endosomal proteinases is a key step in receptor signaling and function and a potential target for therapy.

The endoplasmic reticulum: integration of protein folding, quality control, signaling and degradation.

The endoplasmic reticulum is the entry point into the secretory pathway. To acquire a correct conformation, secretory proteins encounter the endoplasmic reticulum molecular machines of folding, quality control, signaling and disposal, which function as an integrated mechanism. The creation of such a molecular network, spatially regulated, suggests how the endoplasmic reticulum promotes the release of correctly folded secretory proteins.

Specific inhibition by hGRB10zeta of insulin-induced glycogen synthase activation: evidence for a novel signaling pathway.

Grb10 is a member of a family of adapter proteins that binds to tyrosine-phosphorylated receptors including the insulin receptor kinase (IRK). In this study recombinant adenovirus was used to over-express hGrb10zeta, a new Grb10 isoform, in primary rat hepatocytes and the consequences for insulin signaling were evaluated. Over-expression of hGrb10zeta resulted in 50% inhibition of insulin-stimulated IRK autophosphorylation and activation. Analysis of downstream events showed that hGrb10zeta over-expression specifically inhibits insulin-stimulated glycogen synthase (GS) activity and glycogen synthesis without affecting insulin-induced IRS1/2 phosphorylation, PI3-kinase activation, insulin like growth factor binding protein-1 (IGFBP-1) mRNA expression, and ERK1/2 MAP kinase activity. The classical pathway from PI3-kinase through Akt-PKB/GSK-3 leading to GS activation by insulin was also not affected by hGrb10zeta over-expression. These results indicate that hGrb10zeta inhibits a novel and presently unidentified insulin signaling pathway leading to GS activation in liver.

Proteomics characterization of abundant Golgi membrane proteins.

A mass spectrometric analysis of proteins partitioning into Triton X-114 from purified hepatic Golgi apparatus (84% purity by morphometry, 122-fold enrichment over the homogenate for the Golgi marker galactosyl transferase) led to the unambiguous identification of 81 proteins including a novel Golgi-associated protein of 34 kDa (GPP34). The membrane protein complement was resolved by SDS-polyacrylamide gel electrophoresis and subjected to a hierarchical approach using delayed extraction matrix-assisted laser desorption ionization mass spectrometry characterization by peptide mass fingerprinting, tandem mass spectrometry to generate sequence tags, and Edman sequencing of proteins. Major membrane proteins corresponded to known Golgi residents, a Golgi lectin, anterograde cargo, and an abundance of trafficking proteins including KDEL receptors, p24 family members, SNAREs, Rabs, a single ARF-guanine nucleotide exchange factor, and two SCAMPs. Analytical fractionation and gold immunolabeling of proteins in the purified Golgi fraction were used to assess the intra-Golgi and total cellular distribution of GPP34, two SNAREs, SCAMPs, and the trafficking proteins GBF1, BAP31, and alpha(2)P24 identified by the proteomics approach as well as the endoplasmic reticulum contaminant calnexin. Although GPP34 has never previously been identified as a protein, the localization of GPP34 to the Golgi complex, the conservation of GPP34 from yeast to humans, and the cytosolically exposed location of GPP34 predict a role for a novel coat protein in Golgi trafficking.

Tyrosine phosphorylation of p97 regulates transitional endoplasmic reticulum assembly in vitro.

The ATPase associated with different cellular activities family member p97, associated p47, and the t-SNARE syntaxin 5 are necessary for the cell-free reconstitution of transitional endoplasmic reticulum (tER) from starting low-density microsomes. Here, we report that membrane-associated tyrosine kinase and protein-tyrosine phosphatase (PTPase) activities regulate tER assembly by stabilizing (PTPase) or destabilizing (tyrosine kinase) p97 association with membranes. Incubation with the PTPase inhibitor bpV(phen) inhibited tER assembly coincident with the enhanced tyrosine phosphorylation of endogenous p97 and its release from membranes. By contrast, the tyrosine kinase inhibitor, genistein, promoted tER formation and prevented p97 dissociation from membranes while increasing p97 association with the t-SNARE syntaxin 5. Purification of the endogenous tyrosine kinase activity from low-density microsomes led to the identification of JAK-2, whereas PTPH1 was identified as the relevant PTPase. The p97 tyrosine phosphorylation state is proposed to coordinate the assembly of the tER as a regulatory step of the early secretory pathway.

Compartmentalization and insulin-induced translocations of insulin receptor substrates, phosphatidylinositol 3-kinase, and protein kinase B in rat liver.

Physiological doses of insulin in rats resulted in a rapid redistribution of key signaling proteins between subcellular compartments in rat liver. In plasma membranes (PM) and microsomes, insulin induced a rapid decrease in insulin receptor substrate-1/2 (IRS1/2) within 30 sec and an increase in these proteins in endosomes (EN) and cytosol. The level of p85 in PM increased 2.3-fold at 30 sec after insulin stimulation followed by a decrease at 2 min. In this interval, 60-85% and 10-20% of p85 in PM was associated with IRS1 and IRS2, respectively. Thus, in PM, IRS1/2 accounts for almost all of the protein involved in phosphatidylinositol 3-kinase activation. In ENs insulin induced a maximal increase of 40% in p85 recruitment. As in PM, almost all p85 was associated with IRS1/2. The greater level of p85 recruitment to PM was associated with a higher level of insulin-induced recruitment of Akt1 to this compartment (4.0-fold in PM vs. 2.4-fold in EN). There was a close correlation between Akt1 activity and Akt1 phosphorylation at Thr308 and Ser473 in PM and cytosol. However, in ENs the level of Akt1 activity per unit of phosphorylated Akt1 was significantly greater than in PM, indicating that in addition to phosphorylation, another factor(s) modulates Akt1 activation by insulin in rat liver. Our results demonstrate that activation of the insulin receptor kinase and modulation of key components of the insulin signaling cascade occur at the cell surface and within the endosomal system. These data provide further support for the role of the endocytic process in cell signaling.

The heterodimeric structure of glucosidase II is required for its activity, solubility, and localization in vivo.

Glucosidase II is an ER heterodimeric enzyme that cleaves sequentially the two innermost alpha-1,3-linked glucose residues from N-linked oligosaccharides on nascent glycoproteins. This processing allows the binding and release of monoglucosylated (Glc(1)Man(9)GlcNAc(2)) glycoproteins with calnexin and calreticulin, the lectin-like chaperones of the endoplasmic reticulum. We have isolated two cDNA isoforms of the human alpha subunit (alpha1 and alpha2) differing by a 66 bp stretch, and a cDNA for the corresponding beta subunit. The alpha1 and alpha2 forms have distinct mobilities on SDS-PAGE and are expressed in most of the cell lines we have tested, but were absent from the glucosidase II-deficient cell line PHA(R) 2.7. Using COS7 cells, the coexpression of the beta subunit with the catalytic alpha subunit was found to be essential for enzymatic activity, solubilization, and/or stability, and ER retention of the alpha/beta complex. Transfected cell extracts expressing either alpha1 or alpha2 forms with the beta subunit showed similar activities, while mutating( )the nucleophile (D542N) predicted from the glycoside hydrolase Family 31 active site consensus sequence abolished enzymatic activity. In order to compare the kinetic parameters of both alpha1/beta and alpha2/beta forms of human glucosidase II the protein was expressed with the baculovirus expression system. Expression of the human alpha or beta subunit alone led to the formation of active human/insect heteroenzymes, demonstrating functional complementation by the endogenous insect glucosidase II subunits. The activity of both forms of recombinant human glucosidase II was examined with a p-nitrophenyl alpha-D-glucopyranoside substrate, and a two binding site kinetic model for this substrate was shown. The K(M1-2) values and apparent K(i1-2 )for deoxynojirimycin and castanospermine were determined and found to be identical for both isoforms suggesting they have similar catalysis and inhibition characteristics. The substrate specificities of both isoforms using the physiological oligosaccharides were assessed and found to be similar.

Role of p97 and syntaxin 5 in the assembly of transitional endoplasmic reticulum.

Transitional endoplasmic reticulum (tER) consists of confluent rough and smooth endoplasmic reticulum (ER) domains. In a cell-free incubation system, low-density microsomes (1.17 g cc(-1)) isolated from rat liver homogenates reconstitute tER by Mg(2+)GTP- and Mg(2+)ATP-hydrolysis-dependent membrane fusion. The ATPases associated with different cellular activities protein p97 has been identified as the relevant ATPase. The ATP depletion by hexokinase or treatment with either N-ethylmaleimide or anti-p97 prevented assembly of the smooth ER domain of tER. High-salt washing of low-density microsomes inhibited assembly of the smooth ER domain of tER, whereas the readdition of purified p97 with associated p47 promoted reconstitution. The t-SNARE syntaxin 5 was observed within the smooth ER domain of tER, and antisyntaxin 5 abrogated formation of this same membrane compartment. Thus, p97 and syntaxin 5 regulate assembly of the smooth ER domain of tER and hence one of the earliest membrane differentiated components of the secretory pathway.

Association of phosphatidylinositol 3-kinase with the insulin receptor: compartmentation in rat liver.

Phosphatidylinositol 3-kinase (PI 3-kinase) plays an important role in a variety of hormone and growth factor-mediated intracellular signaling cascades and has been implicated in the regulation of a number of metabolic effects of insulin, including glucose transport and glycogen synthase activation. In the present study we have examined 1) the association of PI 3-kinase with the insulin receptor kinase (IRK) in rat liver and 2) the subcellular distribution of PI 3-kinase-IRK interaction. Insulin treatment promoted a rapid and pronounced recruitment of PI 3-kinase to IRKs located at the plasma membrane, whereas no increase in association with endosomal IRKs was observed. In contrast to IRS-1-associated PI 3-kinase activity, association of PI 3-kinase with the plasma membrane IRK did not augment the specific activity of the lipid kinase. With use of the selective PI 3-kinase inhibitor wortmannin, our data suggest that the cell surface IRK beta-subunit is not a substrate for the serine kinase activity of PI 3-kinase. The functional significance for the insulin-stimulated selective recruitment of PI 3-kinase to cell surface IRKs remains to be elucidated.

Cloning and characterization of mammalian UDP-glucose glycoprotein: glucosyltransferase and the development of a specific substrate for this enzyme.

The endoplasmic reticulum enzyme UDP-glucose glycoprotein:glucosyltransferase (UGGT) has the unique property of recognizing incompletely folded glycoproteins and, if they carry an N -linked Man(9)GlcNAc(2)oligosaccharide, of catalyzing the addition of a glucose residue from UDP-glucose. Using peptide sequence information, we have isolated the complete cDNA of rat liver UGGT and expressed it in insect cells. The cDNA specifies an open reading frame which codes for a protein of 1527 residues including an 18 amino acid signal peptide. The protein has a C-terminal tetrapeptide (HEEL) characteristic of endoplasmic reticulum luminal proteins. The purified recombinant enzyme shows the same preference for unfolded polypeptides with N -linked Man(9)GlcNAc(2)glycans as the enzyme purified from rat liver. A genetically engineered Saccharomyces cerevisiae strain capable of producing glyco-proteins with Man(9)GlcNAc(2)core oligosaccharides was constructed and secreted acid phosphatase (G0-AcP) was purified. G0-AcP was used as an acceptor glycoprotein for UGGT and found to be a better substrate than the previously used soybean agglutinin and thyroglobulin. Recombinant rat UGGT has a K (m) of 44 microM for UDP-glucose. A proteolytic fragment of UGGT was found to retain enzymatic activity thus localizing the catalytic site of the enzyme to the C-terminal 37 kDa of the protein. Using site-directed mutagenesis and photoaffinity labeling, we have identified residues D1334, D1336, Q1429, and N1433 to be necessary for the catalytic activity of the enzyme.

The p24 family member p23 is required for early embryonic development.

The p24 family of type I integral-membrane proteins, which are localised in the endoplasmic reticulum (ER), the intermediate compartment and the Golgi apparatus, are thought to function as receptors for cargo exit from the ER and in transport vesicle formation. Members of the p24 family have been found in a molecular complex and are enriched in COPI-coated vesicles, which are involved in membrane traffic between the ER and Golgi complex. Although expressed abundantly, simultaneous deletion of several family members does not appear to affect cell viability and protein secretion in yeast. In order to gain more insights into the physiological roles of different p24 proteins, we generated mice deficient in the expression of one family member, p23 (also called 24delta1, see for alternative nomenclature). In contrast to yeast genetics, in mice disruption of both p23 alleles resulted in early embryonic lethality. Inactivation of one allele led not only to reduced levels of p23 itself but also to reduced levels of other family members. The reduction in steady-state protein levels also induced structural changes in the Golgi apparatus, such as the formation of dilated saccules. The generation of mice deficient in p23 expression has revealed an essential and non-redundant role for p23 in the earliest stages of mammalian development. It has also provided genetic evidence for the participation of p24 family members in oligomeric complexes and indicates a structural role for these proteins in maintaining the integrity of the early secretory pathway.

Calnexin family members as modulators of genetic diseases.

The endoplasmic reticulum (ER) is an intracellular compartment devoted to the synthesis, segregation and folding of soluble and membrane secretory proteins. Some mutations in these proteins lead to their incorrect or incomplete folding in the ER. The ER has a quality control system which detects misfolded proteins and then specifies their fate. Some mutated proteins are retained in the ER wherein they accumulate (Russell bodies for misfolded immunoglobulin heavy chains, the PiZZ for alpha 1-antitrypsin), others are retrotranslocated from the ER and degraded by the cytosolic proteasomal system, and yet other proteins are eventually secreted (in AZC-treated cells). In this review we summarize the role of ER resident proteins in quality control of mutated secretory proteins.

ER/Golgi intermediates acquire Golgi enzymes by brefeldin A-sensitive retrograde transport in vitro.

Secretory proteins exit the ER in transport vesicles that fuse to form vesicular tubular clusters (VTCs) which move along microtubule tracks to the Golgi apparatus. Using the well-characterized in vitro approach to study the properties of Golgi membranes, we determined whether the Golgi enzyme NAGT I is transported to ER/Golgi intermediates. Secretory cargo was arrested at distinct steps of the secretory pathway of a glycosylation mutant cell line, and in vitro complementation of the glycosylation defect was determined. Complementation yield increased after ER exit of secretory cargo and was optimal when transport was blocked at an ER/Golgi intermediate step. The rapid drop of the complementation yield as secretory cargo progresses into the stack suggests that Golgi enzymes are preferentially targeted to ER/Golgi intermediates and not to membranes of the Golgi stack. Two mechanisms for in vitro complementation could be distinguished due to their different sensitivities to brefeldin A (BFA). Transport occurred either by direct fusion of preexisting transport intermediates with ER/Golgi intermediates, or it occurred as a BFA-sensitive and most likely COP I-mediated step. Direct fusion of ER/Golgi intermediates with cisternal membranes of the Golgi stack was not observed under these conditions.

Protein folding in a specialized compartment: the endoplasmic reticulum.

The endoplasmic reticulum ensures proper folding of secretory proteins. In this review, we summarize and discuss the functions of different classes of folding mediators in the secretory pathway and propose updated models of the quality control system.