A substrate preference for the rough endoplasmic reticulum resident protein FKBP22 during collagen biosynthesis.

The biosynthesis of collagens occurs in the rough endoplasmic reticulum and requires a large numbers of molecular chaperones, foldases, and post-translational modification enzymes. Collagens contain a large number of proline residues that are post-translationally modified to 3-hydroxyproline or 4-hydroxyproline, and the rate-limiting step in formation of the triple helix is the cis-trans isomerization of peptidyl-proline bonds. This step is catalyzed by peptidyl-prolyl cis-trans isomerases. There are seven peptidyl-prolyl cis-trans isomerases in the rER, and so far, two of these enzymes, cyclophilin B and FKBP65, have been shown to be involved in collagen biosynthesis. The absence of either cyclophilin B or FKBP65 leads to a recessive form of osteogenesis imperfecta. The absence of FKBP22 leads to a kyphoscoliotic type of Ehlers-Danlos syndrome (EDS), and this type of EDS is classified as EDS type VI, which can also be caused by a deficiency in lysyl-hydroxylase 1. However, the lack of FKBP22 shows a wider spectrum of clinical phenotypes than the absence of lysyl-hydroxylase 1 and additionally includes myopathy, hearing loss, and aortic rupture. Here we show that FKBP22 catalyzes the folding of type III collagen and interacts with type III collagen, type VI collagen, and type X collagen, but not with type I collagen, type II collagen, or type V collagen. These restrictive interactions might help explain the broader phenotype observed in patients that lack FKBP22.

Role of ribosome and translocon complex during folding of influenza hemagglutinin in the endoplasmic reticulum of living cells.

Protein folding in the living cell begins cotranslationally. To analyze how it is influenced by the ribosome and by the translocon complex during translocation into the endoplasmic reticulum, we expressed a mutant influenza hemagglutinin (a type I membrane glycoprotein) with a C-terminal extension. Analysis of the nascent chains by two-dimensional SDS-PAGE showed that ribosome attachment as such had little effect on ectodomain folding or trimer assembly. However, as long as the chains were ribosome bound and inside the translocon complex, formation of disulfides was partially suppressed, trimerization was inhibited, and the protein protected against aggregation.

Acyl-CoA binding protein (ACBP) regulates acyl-CoA:cholesterol acyltransferase (ACAT) in human mononuclear phagocytes.

It is demonstrated that the acyl-CoA:cholesterol acyltransferase (ACAT) enzyme activity in rough endoplasmatic reticulum membranes is regulated by the acyl-CoA binding protein (ACBP). The ACAT activity is strongly inhibited by different ACBP/oleoyl-CoA complexes depending from the molar ratio of protein and fatty acid-CoA. Other lipid binding proteins such as bovine serum albumin and the liver fatty acid binding protein do not show any effects on ACAT activity. In addition, we can show that cholesterol loading with acetylated low density lipoproteins does not lead to an increase of the ACBP mRNA level. Consequently, the increase of the intracellular concentration of fatty acids because of the cholesteryl ester accumulation renders ACAT more active for cholesterol esterification. In binding studies we have characterized binding sites on microsomal membranes for the ACAT substrate oleoyl-CoA and the ACAT inhibitor diazepam. Diazepam competes with oleoyl-CoA and vice versa for its binding to microsomal membranes. This common binding site is suggested to be responsible for the transfer from ACBP-bound oleoyl-CoA to ACAT and, therefore, to be essential for the microsomal cholesterol esterification.

20 S proteasomes are imported as precursor complexes into the nucleus of yeast.

The mechanism by which yeast 20 S proteasomes are imported into the nucleus is still unresolved. Here, we provide the first evidence that 20 S proteasomes are imported as precursor complexes into the nucleus. By using the srp1-49 mutant which is deficient in nuclear import of cargos with classical nuclear localization sequences (cNLS), we show that proteasome precursor complexes associate with importin/karyopherin alphabeta, the cNLS receptor, and that they accumulate inside the cytoplasm. Reconstitution assays revealed that only precursor complexes are targeted to the nuclear envelope (NE) by karyopherin alphabeta. In support, the green fluorescent protein (GFP)-labelled maturation factor Ump1, marking precursor complexes, mainly localizes to the nucleus and around the NE. Our data suggest that nuclear 20 S proteasomes are finally matured inside the nucleus.

An ultrastructural study of cholinergic and non-cholinergic neurons in the laterodorsal and pedunculopontine tegmental nuclei in the rat.

Synaptic connectivity and other ultrastructural features of cholinergic and non-cholinergic neurons in the laterodorsal and pedunculopontine tegmental nuclei were investigated with electron microscopy combined with pre-embedding immunohistochemistry for choline acetyltransferase. Quantitative morphometric analyses were conducted on selected immunopositive as well as immunonegative neurons. The ultrastructure of immunoreactive neurons in the laterodorsal and pedunculopontine tegmental nuclei was similar. In both nuclei, immunoreactive neurons were among the larger neurons, and somatic areas of immunopositive neurons in single thin sections were larger than those of immunonegative neurons by an average of 40%. Immunopositive somata varied in shape, appearing polygonal, fusiform or oval. Regardless of immunoreactivity, however, neurons in the pedunculopontine nucleus tended to have more irregular shapes than those in the laterodorsal tegmental nucleus. Immunoreactive neurons in both the nuclei had abundant cytoplasmic organelles and a large, clear nucleus with a few infoldings. Usually, about a quarter of the surface of an immunopositive soma was covered with astrocytic processes, and some immunopositive somata were directly apposed to an astrocyte. Immunoreactive dendrites and, less frequently, axon terminals were seen in close apposition to endothelial cells of blood capillaries or pericytes. Immunoreactive somata and dendrites in the laterodorsal and pedunculopontine tegmental nuclei received many synapses, mainly from unlabelled axon terminals. The mean number (4.7 +/- 1.8) of synapses received by immunolabelled somata in single thin sections was greater, by about 70%, than those received by unlabelled somata. The presynaptic axon terminals synapsing with immunoreactive somata commonly contained small, round and clear vesicles, and 20% of them contained a few dense-cored vesicles as well. Immunoreactive dendrites, in addition, received synapses from unlabelled axon terminals containing flat and clear vesicles, which accounted for 15% of the synapses with immunoreactive dendrites. Many immunopositive axon terminals were present in both the tegmental nuclei. They contained clear round vesicles, and usually synapsed with unlabelled dendrites. A few immunolabelled axons, however, appeared to synapse with immunopositive somata and dendrites. Immunoreactive fibres were also present in both the tegmental nuclei. They were either thinly myelinated or unmyelinated. In conclusion, the ultrastructural morphology of cholinergic neurons in the laterodorsal and pedunculopontine tegmental nuclei is similar, and these neurons represent a distinct population of neurons in both nuclei in that they are larger and receive more synaptic contacts than non-cholinergic neurons. Cholinergic neurons, however, appear to receive synapses from cholinergic axon terminals only rarely, despite the abundance of cholinergic terminals in the tegmental nuclei.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunocytochemical localization of the prohormone convertases PC1 and PC2 in rat prolactin cells.

The prohormone convertases PC1 and PC2 are subtilisin-related endopeptidases that process prohormone and neuropeptide precursors. Using different ultrastructural immunocytochemical approaches, we have investigated their intracellular distribution in a neuroendocrine cell type that has not been examined thus far, the rat anterior pituitary lactotrope. These cells secrete mainly prolactin and also express the neuroendocrine-specific protein secretogranin II, which is considered a peptide precursor. Our study provides evidence for the expression of PC1 and PC2 in rat lactotropes and provides new information on their subcellular localization. Apart from their presence in the secretory granules, PC1 and PC2 displayed different major localization along the secretory pathway. PC1 immunoreactivity was concentrated in the Golgi apparatus, whereas PC2 immunoreactivity was prominent in the rough endoplasmic reticulum (RER). These observations provide morphological support for previous biochemical analysis of proPC1 and proPC2 post-translational processing, which has demonstrated that PC1 exits very rapidly from the RER, whereas PC2 is retained much longer in this compartment. (J Histochem Cytochem 46:101-108, 1998)

Membrane dipeptidase in the pig exocrine pancreas. Ultrastructural localization and secretion.

The GPI-anchored membrane dipeptidase is the major peptidase activity of the secretory granule membrane in the exocrine pancreas. The enzyme is also found in the granule content and in pancreatic secretions. Immunocytochemical localization confirmed its location in the granule membrane and in the acinar cell apical plasma membrane. In the endoplasmic reticulum and Golgi, membrane dipeptidase was strictly membrane-bound. There was no membrane dipeptidase in duct cells. The release of membrane dipeptidase from the membrane starts in the immature granule. To identify the mechanism responsible for its release, secretions were collected from cannulated conscious pig under basal conditions and atropine perfusion. The latter treatment caused complete inhibition of protein secretion but had a negligible effect on membrane dipeptidase activity in the secretions. In secretions, membrane dipeptidase partitioned into the detergent-rich phase on phase separation in Triton X-114, whereas treatment with bacterial phosphatidylinositol-specific phospholipase C caused the peptidase to partition into the aqueous phase, indicating that the secreted enzyme could come from shedding of membrane fragments at the apical surface or via the action of a previously characterized phospholipase A activity.

Influence of 4-day long treatment with vasoactive intestinal peptide on ultrastructure and function of the rat pinealocytes in organ culture.

Vasoactive intestinal peptide (VIP) is one of neuropeptides involved in the regulation of the pineal gland function. The acute treatment of rat pinealocytes with VIP caused changes in their biochemical parameters. The present study concerns the effects of the chronic treatment with VIP on ultrastructure and function of the rat pinealocytes in organ culture. The pineals of adult male rats were assigned to one of three groups and placed in organ culture for four consecutive days. The pineals of the first group were incubated in the control medium, the pineals of the second group--12 hrs in control medium and 12 hrs in medium with 1 microM VIP (between 20.00 and 8.00) during each day, the pineals of the third group--24 hrs per day in medium with 1 microM VIP. The melatonin concentration was measured using RIA and activity of enzymes using radiochemical methods. Point count method was used in quantitative ultrastructural analysis. Both modes of chronic treatment with VIP increased significantly the level of melatonin secretion during four days of the culture and the content of this hormone in the pineal explants at the end of the experiment. Treatment with the neuropeptide for 12 hrs and 24 hrs per day elevated also the activity of arylalkylamine N-acetyltransferase and hydroxyindole-O-methyltransferase. On the other hand, VIP had no effect on the activity of arylamine-N-acetyltransferase. VIP increased the relative volume of rough endoplasmic reticulum, Golgi apparatus and mitochondria and did not influence the relative volume of lysosomes and lipid droplets as well as the numerical density of dense core vesicles in the examined rat pinealocytes. The obtained results indicate stimulatory effect of chronic treatment with VIP on the synthesis and secretion of melatonin in the rat pinealocytes in vitro. The results of morphological study are in agreement with the obtained biochemical data and point to the increase in secretory and metabolic activity of the rat pinealocytes in response to VIP.

Ultrastructural localization of butyrylcholinesterase in epithelial cells of rat intestine.

The present study reports the histochemical detection, at the ultrastructural level, of butyrylcholinesterase (BChE) in the epithelial cells of rat intestine. The enzyme activity was observed in the crypt cells as well as in the mature cells of the villi. Inside the enterocytes, BChE was seen in the reticulum cisternae, Golgi apparatus and lipid droplets. BChE was also detected in the Grünhagen spaces, associated with chylomicrons. In the intercellular spaces, at the level of the upper part of the cryptic epithelium, the enzyme was found from the base to the apex of the cells. The precise localization of BChE in the cellular structures noted suggests that its function could be related to lipid metabolism and/or cell renewal.

Immuno- and enzyme-cytochemical studies on the pancreatic D-cells in the chicken following vagotomy.

To study the appearance of polymorphic, dense multivesicular body-like structures (MBLS) occurring in the D-cells of pancreatic islets following abdominal vagotomy in the chicken, the D-cells were evaluated by means of quantitative electron microscopy, identified by immunogold labeling and characterized by a method of enzyme cytochemistry. Following vagotomy, secretory granules of the pancreatic D-cells decreased significantly in number, whereas MBLS increased significantly in number. the mean area of the cell, nucleus and mitochondria displayed no significant changes. MBLS were rarely labeled for somatostatin when subjected to immunogold staining. MBLS arranging along the Golgi stacks may possibly be formed in the Golgi apparatus. consequently, it seems unlikely that MBLS are secondary lysosomes revealing crinophagy. Some MBLS showed acid phosphatase (AcPase) activity, although the direct fusion of primary lysosomes exhibiting AcPase activity with MBLS was seldom observed. As a result, it is possible that MBLS may be a kind of modified form of secretory granules and that they may be destroyed by lysosomes under the storing condition, judging from the AcPase reaction.

[NO-ergic function of the Lugaro and Golgi cells in the rabbit cerebellar cortex]. / NO-ergicheskaia funktsiia kletok Liugaro i Gol'dzhi kory mozzhechka krolika.

The distribution of NADPH-diaphorase was studied cytochemically in the rabbit cerebellar cortex. In the granular layer the Golgi cells with positive reaction were found. The highest activity of enzyme was observed in the cytoplasm of intermediate cells of Lugaro, synaptic neurons of Landau, large and middle sized Golgi cells and intercalate cells of Pensa. It is concluded that the Lugaro and Golgi cells may metabolize nitric oxide to be NO-ergic in their mediator specialization.

Partial urethral obstruction enhances NADPH-diaphorase activity in the monkey (Macaca fascicularis) bladder: light and electron microscopic studies.

The effect of partially obstructing the urethra on the nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity in neurons of the intramural ganglia of the monkey (Macaca fascicularis) bladder was examined by light and electron microscopy. Partial urethral ligation was done in adult male monkeys. The animals were sacrificed 2, 4 weeks after partial urethral obstruction. This was compared to controls (normal and sham operated). Urethral obstructed animals were observed to have increased urinary frequency and decreased urinary flow rate. Two weeks after urethral obstruction, the overall NADPH-d activity in the intramural ganglia of the bladder base was enhanced compared to control animals. The frequency of intensely stained NADPH-d positive neurons was increased compared to the control animals. About one-third of intensely stained NADPH-d positive neurons appeared to undergo degenerative changes. At 4 weeks after urethral obstruction, a wide occurrence of NADPH-d positive neurons in advanced stages of degeneration in the bladder base was observed. Cellular debris was strewn among normal looking ganglion cells and along the nerve processes. The proportion of intensely stained NADPH-d positive neurons was relatively lower than the controls. The total number of NADPH-d positive neurons and the nerve fibres in the entire bladder was significantly reduced when compared to control animals. Electron microscopy showed some NADPH-d activity in intramural ganglion cells in 2 weeks after partial urethral obstruction. NADPH-d reaction product (formazan) was deposited on the membranes of the rough endoplasmic reticulum, and the outer membranes of some mitochondria in the intramural neuron. At 4 weeks after urethral obstruction, NADPH-d was present in the membrane of the mitochondria and some mitochondria appeared swollen with disrupted cristae. Present results show that NADPH-d activity in neurons of the intramural ganglia of the monkey (Macaca fascicularis) urinary bladder was increased after two weeks and reduced after 4 weeks of partial urethral obstruction. It is speculated that the increased NADPH-d activity associated with partial urethral obstruction would lead to neuronal damage and death, which may contribute to detrusor overactivity. However, it warrants further investigation to understand the mechanism of neuronal cell death after partial urethral obstruction.

Ribosomes mask cytochrome b5 on rough microsomal vesicles.

Cytochrome b5 is unmasked on the removal of ribosomes by chemical degranulation of rat liver microsomes. Reattachment of ribosomes to stripped membranes remasks this enzyme on the membrane surface. This haemoprotein may be involved either in the attachment of ribosomes to reticular membranes or in protein biosynthesis by membrane-bound ribosomes.

Identification of the ATP transporter of rat liver rough endoplasmic reticulum via photoaffinity labeling and partial purification.

In order to identify the ATP transporter in rat liver rough endoplasmic reticulum (RER), a photoreactive azido derivative of ATP, 3'-O-(p-azidobenzoyl)-ATP (AB-ATP), was synthesized by the reaction of ATP with N-hydroxysuccinimido 4-azidobenzoate (NHS-AB). The activity of the ATP transporter was determined by measuring the influx of [8-14C]ATP. The ATP transport had an apparent Km value of 6.5 microM and a Vmax of 1 nmol min-1 (mg of protein)-1. The transport of ATP was specifically inhibited by AB-ATP and 4, 4'-diisothiocyanatostilbene-2', 2'-disulfonic acid (DIDS). Under a dim light, AB-ATP was a competitive inhibitor of the ATP transport with Ki value of 0.19 microM, which indicates that AB-ATP has a high affinity for the ATP transporter, so it can be utilized as a photoaffinity probe for the identification of the ATP transporter in rat liver RER. An SDS--PAGE analysis of RER vesicles photolabeled with [gamma-32P]AB-ATP indicates the presence of a 56-kDa protein. The 56-kDa protein was completely protected from photoaffinity labeling by 10 microM ATP but not by 30 microM GTP. The specific labeling of the 56-kDa protein was sensitive to the anion transport inhibitor DIDS. In order to confirm whether the apparent uptake of ATP was due to the 56-kDa protein, the ATP transporter was partially purified through two successive ion-exchange chromatography steps (DEAE and Mono-S). The fraction showing the high activity of the ATP transporter also contained the 56-kDa protein photolabeled with [gamma-32P]AB-ATP. On the basis of the photoaffinity labeling and reconstitution experiment, we conclude that the 56-kDa protein represents the ATP transporter in rat liver RER.

Light and electron microscopic studies of the distribution of NADPH-diaphorase in the rat upper thoracic spinal cord with special reference to the spinal autonomic region.

The distribution of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) was examined in the upper thoracic segment of the spinal cord in rat. Under the light microscope, NADPH-d positive cell bodies and fibers were readily detected in the following areas: 1) the dorsal horn; 2) the dorsolateral funiculus and lateral spinal neurons; 3) spinal autonomic region, consisting of the nucl. intermediolaterialis pars funicularis, nucl. intermediolateralis pars principalis, nucl. intercalatus spinalis and nucl. intercalatus pars paraependymalis; and 4) in the white matter lateral to the nucl. intermediolateralis pars funicularis. In the nucl. intermediolateralis pars principalis, the positive dendrites, running in bundles, were directed medially in the gray matter towards the central canal as well as laterally in the white matter towards the pia mater. The medially-directed positive dendrites fomed a subependymal plexus around the central canal. A dense bundle of NADPH-d positive fibers were also observed running longitudinally. Combined retrograde tracing with fluorogold and NADPH-d histochemistry study revealed that some of the NADPH-d positive neurons, due to their fluorescence labelling, were sympathetic preganglionic neurons that innervated the superior cervical ganglion. Under the electron microscope, the reaction products in the neurons of the nucl. intermediolateralis pars principalis were deposited in their nuclear envelope, rough endoplasmic reticulum, mitochondria and Golgi apparatus. In the neuropil, three types of synaptic configurations were observed: between NADPH-d negative axon terminals and NADPH-d positive dendrites, between NADPH-d positive axon terminals and NADPH-d negative dendrites, and between NADPH-d positive axons terminals and NADPH-d positive dendrites. These synaptic configurations suggest that the neurons are regulated by nitric oxide released from both pre- and post-synaptic elements. The sources of the NADPH-d positive axon terminals associated with the neurons remain unclear although the possibility of their being derived from supraspinal origins has to be considered. The ultrastructural demonstration of NADPH-d reaction product in the three major types of glial cells suggests that nitric oxide might be produced by these cells, but its functional significance awaits further investigation.

Endoplasmic reticulum kifunensine-resistant alpha-mannosidase is enzymatically and immunologically related to the cytosolic alpha-mannosidase.

Studies were undertaken to evaluate the relationship of the recently described (S. Weng and R. G. Spiro, 1993, J. Biol. chem. 268, 25656-25663) rat liver kifunensine (KIF)-resistant mannosidase (ER mannosidase II) to the mannose-trimming enzyme of cytosol. We observed that the ER mannosidase II manifests a large number of catalytic and immunological properties similar to those of the cytosolic alpha-mannosidase, which contrast with the quite different characteristics of the KIF-sensitive enzyme (ER mannosidase I). In addition to a mutual resistance to KIF inhibition, the cytosolic enzyme and ER mannosidase II have comparable susceptibility to blocking by swainsonine and 1,4-dideoxy-1,4-imino-D-mannitol, and the latter agent was found to function effectively both in vitro and in vivo. The cytosolic and ER II mannosidases were alike in specifically excising the terminal mannose of the alpha 1,6-linked chain of Man9GlcNAc to yield Man8GlcNAc isomer C; in preferentially hydrolyzing polymannose-GlcNAc1 over polymannose-GlcNAc2 substrates; and in cleaving p-nitrophenyl alpha-D-mannoside. An immunological cross-reactivity between cytosolic mannosidase (M(r) 105 kDa) and ER mannosidase II (M(r) 82 kDa), neither of which is N-glycosylated, was established, suggesting that the latter is translocated posttranslationally into the lumen of the ER compartment in which we found it to be present as a soluble protein. Since antibodies directed against a sequence near the C-terminal end of the cytosolic enzyme reacted with ER mannosidase II while those against a sequence close to the N-terminus did not, it is likely that a proteolytic cleavage of the latter segment takes place during or after translocation. The absence in ER mannosidase II of the pronounced cobalt activation of the cytosolic enzyme suggests that the portion of the polypeptide chain removed during the 105- to 82-kDa conversion includes the binding domain for this ion.

Markers for trans-Golgi membranes and the intermediate compartment localize to induced membranes with distinct replication functions in flavivirus-infected cells.

Replication of the flavivirus Kunjin virus is associated with virus-induced membrane structures within the cytoplasm of infected cells; these membranes appear as packets of vesicles associated with the sites of viral RNA synthesis and as convoluted membranes (CM) and paracrystalline arrays (PC) containing the components of the virus-specified protease (E. G. Westaway, J. M. Mackenzie, M. T. Kenney, M. K. Jones, and A. A. Khromykh, J. Virol. 71:6650-6661, 1997). To determine the cellular origins of these membrane structures, we compared the immunolabelling patterns of several cell markers in relation to these sites by immunofluorescence and immunoelectron microscopy. A marker for the trans-Golgi membranes and the trans-Golgi network, 1,4-galactosyltransferase (GalT), was redistributed to large foci in the cytoplasm of Kunjin virus-infected cells, partially coincident with immunofluorescent foci associated with the putative sites of viral RNA synthesis. As determined by immunoelectron microscopy, the induced vesicle packets contained GalT, whereas the CM and PC contained a specific protein marker for the intermediate compartment (ERGIC53). A further indicator of the role of cellular organelles in their biogenesis was the observation that the Golgi apparatus-disrupting agent brefeldin A prevented further development of immunofluorescent foci of induced membranes if added before the end of the latent period but that once formed, these membrane foci were resistant to brefeldin A dispersion. Reticulum membranes emanating from the induced CM and PC were also labelled with the rough endoplasmic reticulum marker anti-protein disulfide isomerase and were obviously redistributed during infection. This is the first report identifying trans-Golgi membranes and the intermediate compartment as the apparent sources of the flavivirus-induced membranes involved in events of replication.

GFP-labelling of 26S proteasomes in living yeast: insight into proteasomal functions at the nuclear envelope/rough ER.

26S proteasomes are multisubunit protease complexes that play the central role in the ubiquitin-dependent protein degradation pathway. The proteolytically active core is formed by the 20S proteasome. Regulatory subunits, principally the 19S cap complex, confer the specificity towards ubiquitinated substrates and an ATP-dependence on proteolysis. Green fluorescence protein (GFP)-tagged versions of either an alpha-subunit of the 20S core or an ATPase subunit of the 19S cap complex were functionally incorporated into the protease complex, thus allowing to monitor the subcellular distribution of 26S proteasomes in living yeast. Our localization studies suggest that proteasomal proteolysis mainly occurs at the nuclear envelope (NE)/rough ER. Implications of proteasomal functions at the NE/rough ER are discussed in the context of published work on ER degradation and with regard to possible targeting mechanisms.