A novel dynamin-like protein associates with cytoplasmic vesicles and tubules of the endoplasmic reticulum in mammalian cells

Dynamins are 100-kilodalton guanosine triphosphatases that participate in the formation of nascent vesicles during endocytosis. Here, we have tested if novel dynamin-like proteins are expressed in mammalian cells to support vesicle trafficking processes at cytoplasmic sites distinct from the plasma membrane. Immunological and molecular biological methods were used to isolate a cDNA clone encoding an 80-kilodalton novel dynamin-like protein, DLP1, that shares up to 42% homology with other dynamin-related proteins. DLP1 is expressed in all tissues examined and contains two alternatively spliced regions that are differentially expressed in a tissue-specific manner. DLP1 is enriched in subcellular membrane fractions of cytoplasmic vesicles and endoplasmic reticulum. Morphological studies of DLP1 in cultured cells using either a specific antibody or an expressed green fluorescent protein (GFP)- DLP1 fusion protein revealed that DLP1 associates with punctate cytoplasmic vesicles that do not colocalize with conventional dynamin, clathrin, or endocytic ligands. Remarkably, DLP1-positive structures coalign with microtubules and, most strikingly, with endoplasmic reticulum tubules as verified by double labeling with antibodies to calnexin and Rab1 as well as by immunoelectron microscopy. These observations provide the first evidence that a novel dynamin-like protein is expressed in mammalian cells where it associates with a secretory, rather than endocytic membrane compartment.     

Rapid cytochrome c release, activation of caspases 3, 6, 7 and 8 followed by Bap31 cleavage in HeLa cells treated with photodynamic therapy

Photodynamic therapy (PDT) is a clinical approach that utilizes light-activated drugs for the treatment of a variety of pathologic conditions. The initiating events of PDT-induced apoptosis are poorly defined. It has been shown for other proapoptotic stimuli that the integral endoplasmic reticulum protein Bap31 is cleaved by caspases 1 and 8, but not by caspase-3. Further, a 20 kDa Bap31 cleavage fragment is generated which can induce apoptosis. In the current report, we sought to determine whether Bap31 cleavage and generation of p20 is an early event in PDT-induced apoptosis. The mitochondrial release of cytochrome c, involvement of caspases 1, 2, 3, 4, 6, 7, 8, and 10 and the status of several known caspase substrates, including Bap31, were evaluated in PDT-treated HeLa cells. Cytochrome c appeared in the cytosol immediately following light activation of the photosensitizer benzoporphyrin derivative monoacid ring A. Activation of caspases 3, 6, 7, and 8 was evident within 1-2 h post PDT. Processing of caspases 1, 2, 4, and 10 was not observed. Cleavage of Bap31 was observed at 2-3 h post PDT. The caspase-3 inhibitor DEVD-fmk blocked caspase-8 and Bap31 cleavage suggesting that caspase-8 and Bap31 processing occur downstream of caspase-3 activation in PDT-induced apoptosis. These results demonstrate that release of mitochondrial cytochrome c into the cytoplasm is a primary event following PDT, preceding caspase activation and cleavage of Bap31. To our knowledge, this is the first example of a chemotherapeutic agent inducing caspase-8 activation and demonstrates that caspase-8 activation can occur after cytochrome c release.     

Hepatitis C virus glycoprotein complex localization in the endoplasmic reticulum involves a determinant for retention and not retrieval

The hepatitis C virus (HCV) genome encodes two envelope glycoproteins (E1 and E2). These glycoproteins interact to form a noncovalent heterodimeric complex which in the cell accumulates in endoplasmic reticulum (ER)-like structures. The transmembrane domain of E2, at least, is involved in HCV glycoprotein complex localization in this compartment. In principle, ER localization of a protein can be the consequence of actual retention in this organelle or of retrieval from the Golgi. To determine which of these two mechanisms is responsible for HCV glycoprotein complex accumulation in the ER, the precise localization of these proteins was studied by immunofluorescence, and the processing of their glycans was analyzed. Immunolocalization of HCV glycoproteins after nocodazole treatment suggested an ER retention. In addition, HCV glycoprotein glycans were not modified by Golgi enzymes, indicating that the ER localization of these proteins is not because of their retrieval from the cis Golgi. Retention of HCV glycoprotein complexes in the ER without retrieval suggests that this compartment plays an important role for the acquisition of the envelope of HCV particles. A true retention in the ER was also observed for E2 expressed in the absence of E1 or for a chimeric protein containing the ectodomain of CD4 in fusion with the transmembrane domain of E2. These data indicate that, in HCV glycoprotein complex, the transmembrane domain of E2, at least, is responsible for true retention in the ER, without recycling through the Golgi.     

Role of xklp3, a subunit of the Xenopus kinesin II heterotrimeric complex, in membrane transport between the endoplasmic reticulum and the Golgi apparatus

The function of the Golgi apparatus is to modify proteins and lipids synthesized in the ER and sort them to their final destination. The steady-state size and function of the Golgi apparatus is maintained through the recycling of some components back to the ER. Several lines of evidence indicate that the spatial segregation between the ER and the Golgi apparatus as well as trafficking between these two compartments require both microtubules and motors. We have cloned and characterized a new Xenopus kinesin like protein, Xklp3, a subunit of the heterotrimeric Kinesin II. By immunofluorescence it is found in the Golgi region. A more detailed analysis by EM shows that it is associated with a subset of membranes that contain the KDEL receptor and are localized between the ER and Golgi apparatus. An association of Xklp3 with the recycling compartment is further supported by a biochemical analysis and the behavior of Xklp3 in BFA-treated cells. The function of Xklp3 was analyzed by transfecting cells with a dominant-negative form lacking the motor domain. In these cells, the normal delivery of newly synthesized proteins to the Golgi apparatus is blocked. Taken together, these results indicate that Xklp3 is involved in the transport of tubular-vesicular elements between the ER and the Golgi apparatus.     

Interactions between newly synthesized glycoproteins, calnexin and a network of resident chaperones in the endoplasmic reticulum

Calnexin is a membrane-bound lectin and a molecular chaperone that binds newly synthesized glycoproteins in the endoplasmic reticulum (ER). To analyze the oligomeric properties of calnexin and calnexin-substrate complexes, sucrose velocity gradient centrifugation and chemical cross-linking were used. After CHAPS solubilization of Chinese Hamster Ovary cells, the unoccupied calnexin behaved as a monomer sedimenting at 3.5 S20,W. For calnexin-substrate complexes the S-values ranged between 3.5-8 S20,W, the size increasing with the molecular weight of the substrate. Influenza hemagglutinin, a well-characterized substrate associated with calnexin in complexes that sedimented at 5-5.5 S20,W. The majority of stable complexes extracted from cells, appeared to contain a single calnexin and a single substrate molecule, with about one third of the calnexin in the cell being unoccupied or present in weak associations. However, when chemical cross-linking was performed in intact cells, the calnexin-substrate complexes and calnexin itself was found to be part of a much larger heterogeneous protein network that included other ER proteins. Pulse-chase analysis of influenza-infected cells combined with chemical cross-linking showed that HA was part of large, heterogeneous, cross-linked entities during the early phases of folding, but no longer after homotrimer assembly. The network of weakly associated resident ER chaperones which included BiP, GRP94, calreticulin, calnexin, and other proteins, may serve as a matrix that binds early folding and assembly intermediates and restricts their exit from the ER.     

Interaction of polycyclic aromatic hydrocarbons and flavones with cytochromes P450 in the endoplasmic reticulum: effect on CO binding kinetics

The flash photolysis technique was used to examine the kinetics of CO binding to cytochromes P450 in rat liver microsomes. The effect of polycyclic aromatic hydrocarbons (PAHs) and flavones was used to distinguish the kinetic behavior of the PAH-metabolizing P450 1A1 from that of the remaining multiple microsomal P450s. Applying this approach to microsomes from 3-methylcholanthrene-treated rats showed that although all tested PAHs accelerated CO binding to P450 1A1, the extent varied markedly for different PAHs. The tricyclic PAHs phenanthrene and anthracene enhanced CO binding by 37- and 49-fold, respectively, while several tetracyclic and pentacyclic PAHs increased the rate by 3-16-fold. The results indicate that PAHs exert a dual effect on the rate of CO binding to P450 1A1: a general enhancement via widening of the CO access channel and a reduction that is dependent on PAH size. Although 5,6-benzoflavone increased the rate of CO binding to P450 1A1 by 3.5-fold, it additionally decelerated binding to a constitutive P450 by 15-fold. This flavone thus exerts markedly different effects on two P450s within the same microsomal sample. In contrast, the sole effect of 7,8-benzoflavone was acceleration of CO binding to P450 1A1 by 18-fold. The divergent effects of these isomeric flavones, which only differ in positioning of an aromatic ring, illustrate the sensitivity of CO binding to substrate structure. The varying effects of these PAHs and flavones on CO binding kinetics show that they differentially modulate P450 conformation and access of ligands to the P450 heme and demonstrate that binding of carcinogens to a specific target P450 can be evaluated in its native microsomal milieu.     

PIG-B, a membrane protein of the endoplasmic reticulum with a large lumenal domain, is involved in transferring the third mannose of the GPI anchor

Many eukaryotic cell surface proteins are bound to the membrane via the glycosylphosphatidylinositol (GPI) anchor that is covalently linked to their carboxy-terminus. The GPI anchor precursor is synthesized in the endoplasmic reticulum (ER) and post-translationally linked to protein. We cloned a human gene termed PIG-B (phosphatidylinositol glycan of complementation class B) that is involved in transferring the third mannose. PIG-B encodes a 554 amino acid, ER transmembrane protein with an amino-terminal portion of approximately 60 amino acids on the cytoplasmic side and a large carboxy-terminal portion of 470 amino acids within the ER lumen. A mutant PIG-B lacking the cytoplasmic portion remains active, indicating that the functional site of PIG-B resides on the lumenal side of the ER membrane. The PIG-B gene was localized to chromosome 15 at q21-q22. This autosomal location would explain why PIG-B is not involved in the defective GPI anchor synthesis in paroxysmal nocturnal hemoglobinuria, which is always caused by a somatic mutation of the X-linked PIG-A gene.     

Demonstration that a kifunensine-resistant alpha-mannosidase with a unique processing action on N-linked oligosaccharides occurs in rat liver endoplasmic reticulum and various cultured cells

A novel alpha-mannosidase has been identified in rat liver endoplasmic reticulum (ER) which at neutral pH processes the Man9GlcNAc oligosaccharide of glycoproteins by specifically cleaving the terminal mannose residue of the alpha 1,6-linked chain to yield Man8GlcNAc, isomer C. This enzyme accounted for about half of the total ER alpha-mannosidase activity and was fully active at the concentration (0.25 microM) of kifunensine (KIF) completely inhibitory to the action of the ER enzyme which by removing the terminal sugar of the middle chain converts Man9GlcNAc to Man8GlcNAc isomer B; both ER enzymes, however, were inhibited in a similar manner by 1-deoxymannojirimycin (IC50 = 0.2 mM) and their action could not be distinguished with this agent. The KIF-resistant mannosidase which functioned optimally in the presence of 0.1-0.5% Triton X-100 did not show the high susceptibility to EDTA demonstrated by the KIF-sensitive enzyme and unlike the latter had the capacity to hydrolyze p-nitrophenyl-alpha-D-mannoside (Km = 0.45 mM); it had no specific cation requirements, but its activity was greatly reduced in the presence of Zn2+. In isolated ER membranes as well as in intact carbonyl cyanide m-chlorophenylhydrazone-treated cells, the processing pattern was substantially different in the presence of KIF than in its absence; while in the latter instance Man9GlcNAc was readily converted to Man6GlcNAc, the KIF-resistant enzyme was limited in its capacity to go beyond Man8GlcNAc. The KIF-resistant alpha-mannosidase was found in substantial amounts in all cell lines examined (HL-60, BW5147.3, MOLT-4, K-562, HepG2, Chinese hamster ovary, F-9, Madin-Darby canine kidney, FRTL-5). The finding that mannose removal from N-linked oligosaccharides can be initiated in two distinctive manners substantially broadens our concept of the processing events which can occur before a glycoprotein reaches the Golgi complex or to which ER resident molecules can be exposed.     

Mtd, a novel Bcl-2 family member activates apoptosis in the absence of heterodimerization with Bcl-2 and Bcl-XL

We have identified and characterized Mtd, a novel regulator of apoptosis. Sequence analysis revealed that Mtd is a member of the Bcl-2 family of proteins containing conserved BH1, BH2, BH3, and BH4 regions and a carboxyl-terminal hydrophobic domain. In adult tissues, Mtd mRNA was predominantly detected in the brain, liver, and lymphoid tissues, while in the embryo Mtd mRNA was detected in the liver, thymus, lung, and intestinal epithelium. Expression of Mtd promoted the death of primary sensory neurons, 293T cells and HeLa cells, indicating that Mtd is a proapoptotic protein. Unlike all other known death agonists of the Bcl-2 family, Mtd did not bind significantly to the survival-promoting proteins Bcl-2 or Bcl-XL. Furthermore, apoptosis induced by Mtd was not inhibited by Bcl-2 or Bcl-XL. A Mtd mutant with glutamine substitutions of highly conserved amino acids in the BH3 domain retained its ability to promote apoptosis, further indicating that Mtd does not promote apoptosis by heterodimerizing with Bcl-2 or Bcl-XL. Mtd-induced apoptosis was not blocked by broad range synthetic caspase inhibitors z-VAD-fmk or a viral protein CrmA. Mtd is the first example of a naturally occurring Bcl-2 family member that can activate apoptosis independently of heterodimerization with survival-promoting Bcl-2 and Bcl-XL.     

BiP, a major chaperone protein of the endoplasmic reticulum lumen, plays a direct and important role in the storage of the rapidly exchanging pool of Ca2+

The activity of BiP, the major chaperone of the endoplasmic reticulum (ER) lumen, is known to be Ca2+-regulated; however, the participation of this protein in the ER storage of the cation has not yet been investigated. Here such a role is demonstrated in human epithelial (HeLa) cells transiently transfected with the hamster BiP cDNA and incubated in Ca2+-free medium, as revealed by two different techniques. In the first, co-transfected aequorin was employed as a probe for assaying either the cytosolic of the mitochondrial free Ca2+ concentration. By this approach higher Ca2+ release responses were revealed in BiP-transfected cells by experiments in which extensive store depletion was induced either by repetitive stimulation with inositol 1,4,5-trisphosphate-generating agonists or by treatment with the Ca2+ ionophore, A23187. In the second technique the cells were loaded at the equilibrium with 45Ca, and the release of the tracer observed upon treatment with thapsigargin, a blocker of the ER Ca2+ ATPases, was larger in BiP-transfected than in control cells. The latter results were obtained also when BiP was overexpressed not via transfection but as a response to ER stress by tunicamycin. These results are sustained by increases of the ER Ca2+ storage capacity rather than by artifacts or indirect readjustments induced in the cells by the overexpression of the chaperone since (a) the exogenous and endogenous BiP were both confined to the ER, (b) the expression levels of other proteins active in the ER Ca2+ storage were not changed, and (c) effects similar to those of wild type BiP were obtained with a deletion mutant devoid of chaperone activity. The specificity of the results was confirmed by parallel 45Ca experiments carried out in HeLa cells transfected with two other Ca2+-binding proteins, calreticulin and CaBP2(ERp72), only the first of which induced increases of Ca2+ capacity. We conclude that BiP has a dual function, in addition to its chaperone role it is a bona fide ER lumenal Ca2+ storage protein contributing, under resting cell conditions, to around 25% of the store, with a stoichiometry of 1-2 moles of calcium/mole of BiP.     

Dhh1p, a putative RNA helicase, associates with the general transcription factors Pop2p and Ccr4p from Saccharomyces cerevisiae

It is generally believed that the neuronal form of nitric oxide synthase (nNOS) is constitutively expressed and that regulation of this enzyme's activity is mediated solely by changes in cytosolic calcium concentration. Serendipitously, however, we observed that pretreatment of Chinese hamster ovary (CHO) cells, which coexpress muscarinic M1 receptors and nNOS, with 3.3 microM or 1 mM carbachol (CCh) for 48 h resulted in marked enhancement of maximal muscarinic receptor-stimulated nNOS activity as determined by L-[3H]citrulline and cyclic [3H]GMP production. This was accompanied by a decrease in the potency of CCh. Muscarinic receptor density was reduced in the agonist-pretreated cells, as determined by specific [N-methyl-3H]scopolamine methyl chloride binding, whereas competition binding studies revealed no changes in agonist affinity. Both receptor-stimulated inositol phosphate formation and elevation of intracellular calcium concentrations were found to be desensitized in agonist-pretreated cells in a manner dependent on CCh pretreatment concentration. It is interesting that ionomycin-stimulated nNOS activity was greater in CCh-pretreated cells. Also, western analysis revealed increased nNOS immunoreactivity in pretreated cells. A similar increase in nNOS immunoreactivity following agonist treatment was demonstrated in N1E-115 neuroblastoma cells, which endogenously express nNOS and muscarinic M1 receptors. Thus, the enhancement of maximal receptor-stimulated nNOS activity following agonist pretreatment can be attributed to up-regulation of nNOS. It is interesting that this augmentation of the response takes place in spite of receptor down-regulation and desensitization of multiple steps involved in nNOS activation.     

The Hsp70 homologue Lhs1p is involved in a novel function of the yeast endoplasmic reticulum, refolding and stabilization of heat-denatured protein aggregates

Heat stress is an obvious hazard, and mechanisms to recover from thermal damage, largely unknown as of yet, have evolved in all organisms. We have recently shown that a marker protein in the ER of Saccharomyces cerevisiae, denatured by exposure of cells to 50 degrees C after preconditioning at 37 degrees C, was reactivated by an ATP-dependent machinery, when the cells were returned to physiological temperature 24 degrees C. Here we show that refolding of the marker enzyme Hsp150Delta-beta-lactamase, inactivated and aggregated by the 50 degrees C treatment, required a novel ER-located homologue of the Hsp70 family, Lhs1p. In the absence of Lhs1p, Hsp150Delta-beta-lactamase failed to be solubilized and reactivated and was slowly degraded. Coimmunoprecipitation experiments suggested that Lhs1p was somehow associated with heat-denatured Hsp150Delta- beta-lactamase, whereas no association with native marker protein molecules could be detected. Similar findings were obtained for a natural glycoprotein of S. cerevisiae, pro-carboxypeptidase Y (pro-CPY). Lhs1p had no significant role in folding or secretion of newly synthesized Hsp150Delta-beta-lactamase or pro-CPY, suggesting that the machinery repairing heat-damaged proteins may have specific features as compared to chaperones assisting de novo folding. After preconditioning and 50 degrees C treatment, cells lacking Lhs1p remained capable of protein synthesis and secretion for several hours at 24 degrees C, but only 10% were able to form colonies, as compared to wild-type cells. We suggest that Lhs1p is involved in a novel function operating in the yeast ER, refolding and stabilization against proteolysis of heatdenatured protein. Lhs1p may be part of a fundamental heat-resistant survival machinery needed for recovery of yeast cells from severe heat stress.     

Electromyography of the reticulum, abomasum and duodenum in dairy cows with left displacement of the abomasum

Electromyographic (EMG) recordings of the reticulum, abomasal corpus, pyloric antrum and duodenum of six dairy cows with left displacement of the abomasum (LDA) were made in order to substantiate abomasal atony as a prerequisite to abomasal displacement. EMG recordings were made when LDA was present as well as when absent. Mean values were determined in five of six cows for the maximum peak or amplitude, mean peak values, peak-to-peak interval and count of the electrical response activity (ERA) for each 15 min segment of the waveform recordings. Segments containing phase III migrating myoelectric activity were not analysed. LDA positive periods were compared to LDA negative periods in each cow. The 6 h period (transition period) prior to the diagnosis of LDA was analysed separately. Paired t-tests were applied to group values with statistical significance established at the P = 0.05 level. There was a significant decrease in the ERA count during the LDA positive periods in the abomasal corpus (-1.40% to -7.88%, P = 0.0217) and in the pyloric antrum (-2.05% to -11.98%, P = 0.0430). A corresponding significant increase occurred in the peak-to-peak interval. During the transition period spike activity in the duodenum increased 0.5% to 48.31% (P = 0.0474) and the peak-to-peak interval was significantly decreased. No extended periods of atony were observed in the abomasum during this study.