Distribution of covalently bound and non-covalently bound secretory component on sbuclasses of rabbit secretory IgA.

The distribution of non-covalently bound secretory component (SC) on the two subclasses, IgA-f and IgA-g of rabbit secretory IgA (sIgA) was determined; the two subclasses were separated from each other by the use of antibody-immunosorbent columns and were subjected to SDS polyacrylamide gel electrophoresis. No SC appeared to be dissociated from the IgA-f molecules from each of 11 different rabbits; the IgA-g molecules, however, did have SC which was dissociated by SDS. Thus, all of the noncovalently bound SC on rabbit sIgA resides on the IgA-g subclass molecules.     

Isolation of a protein from the plasma membrane of adrenal medulla which binds to secretory vesicles.

Solubilized proteins of the plasma membrane of bovine adrenal medulla were fractionated on the basis of their affinity for secretory vesicles. The isolation procedure included preparation of a highly purified fraction of plasma membranes, its solubilization in detergent, and application to a column prepared from glutaraldehyde-fixed chromaffin granules. Using this technique, one major polypeptide (80% of the material bound) was isolated. This protein has been shown to originate from the plasma membrane and has no affinity for fixed bovine adrenal medullary mitochondria or lysosomes. It is eluted most effectively by low pH (3.0) and can be rebound and re-eluted from fixed secretory granules. In sodium dodecyl sulfate and beta-mercaptoethanol it has an apparent molecular weight of 51,000. In addition, two minor components, comprising about 20% of the material bound were detected having apparent molecular weights in sodium dodecyl sulfate of 14,000 and 62,000. It is suggested that such a molecule could function as a plasma membrane-located receptor for chromaffin granules during the secretory process.     

A bovine epithelial membrane protein that binds polymeric immunoglobulin and has a structure related to that of bovine secretory component.

A protein of Mr 94000 was isolated from detergent-solubilized bovine intestinal, liver and mammary-gland membranes. It binds immunoglobulin M and also undergoes proteolytic fragmentation in a similar manner to bovine secretory component (Mr 74000). The affinity-purified membrane protein is therefore most likely to be the bovine epithelial receptor for polymeric immunoglobulin. A structural model is proposed.     

A ras effector domain mutant which is temperature sensitive for cellular transformation: interactions with GTPase-activating protein and NF-1.

A series of v-rasH effector domain mutants were analyzed for their ability to transform rat 2 cells at either low or high temperatures. Three mutants were found to be significantly temperature sensitive: Ile-36 changed to Leu, Ser-39 changed to Cys (S39C), and Arg-41 changed to Leu. Of these, the codon 39 mutant (S39C) showed the greatest degree of temperature sensitivity. When the same mutation was analyzed in the proto-oncogene form of ras(c-rasH), this gene was also found to be temperature sensitive for transformation. Biochemical analysis of the proteins encoded by v-rasH(S39C) and c-rasH(S39C) demonstrated that the encoded p21ras proteins were stable and bound guanine nucleotides in vivo at permissive and nonpermissive temperatures. On the basis of these findings, it is likely that the temperature-sensitive phenotype results from an inability of the mutant (S39C) p21ras to interact properly with the ras target effector molecule(s) at the nonpermissive temperature. We therefore analyzed the interaction between the c-rasH(S39C) protein and the potential target molecules GTPase-activating protein (GAP) and the GAP-related domain of NF-1, on the basis of stimulation of the mutant p21ras GTPase activity by these molecules in vitro. Assays conducted across a range of temperatures revealed no temperature sensitivity for stimulation of the mutant protein, compared with that of authentic c-rasH protein. We conclude that for this mutant, there is a dissociation between the stimulation of p21ras GTPase activity by GAP and the GAP-related domain NF-1 and their potential target function. Our results are also consistent with the existence of a distinct, as-yet-unidentified effector for mammalian ras proteins.     

A microtubule-associated protein (MAP1) which is expressed at elevated levels during development of the rat cerebellum.

Monoclonal antibody (MAb) G10 labels a single high mol. wt. (HMW) band on Western blots of microtubule preparations from 2 day old rat brain. The G10 antigen is thermolabile and co-migrates with microtubule-associated protein (MAP)1 from young rat brain on low percentage (5%) polyacrylamide-SDS gels. The G10 antigen decreases by about five times from birth to adulthood in the rat cerebellum. The same single band is labelled on Western blots of homogenates of whole neonatal rat brain but no labelling is found using neonatal or adult kidney, lung or liver. We have therefore identified a brain-specific MAP1, designated MAP1(x). Immunofluorescence microscopy using MAb G10 on parasagittal sections of rat cerebella shows labelling of the newly formed molecular layer in 6 day old rats. Only a narrow band close to the pial surface is labelled in 18 day old animals, which disappears in the adult. Labelling of the cerebellar white matter found in young rats also disappears. Neurones but not flat cells in cerebellar cultures label with MAb G10. All staining patterns are consistent with an axonal distribution of the antigen. MAP1(x) may be part of a developmentally regulated microtubule structure.     

Detection of IgA and IgM antibodies to HIV-1 in neonates by radioimmune western blotting.

OBJECTIVE--To detect infection with HIV-1 by IgA and IgM response at birth in children born to HIV-1 seropositive mothers. DESIGN--Western blotting and radioimmune western blotting on stored sera from infected and uninfected babies born to HIV-1 seropositive mothers. Sera were pretreated to remove IgG. SETTING--Parma and Bologna, Italy. SUBJECTS--12 infected and five uninfected babies born to HIV-1 seropositive mothers and three babies born to seronegative mothers. MAIN OUTCOME MEASURES--Effectiveness of western blotting and radioimmune western blotting in detecting antibodies to HIV-1 gene products. RESULTS--With conventional western blotting we found IgA class antibodies to HIV-1 proteins in serum from three out of 12 infected children; in two of these three the serum was collected at age 3 months (positive controls). Radioimmune western blotting detected both IgA and IgM antibodies in serum from all infected children tested, whereas all serum from uninfected children born to seropositive and seronegative mothers showed no such antibodies. CONCLUSION--Although the technique should be tested on more patients, radioimmune western blotting seems to be a valuable tool for serological diagnosis of congenital HIV-1 infection at birth in neonates born to seropositive mothers.     

Post-translational regulation of IgM expression in B lymphocytes. Selective nonlysosomal degradation of assembled secretory IgM is temperature-dependent and occurs prior to the trans-Golgi.

B cells express on their surface the membrane form of IgM (mIgM). Upon differentiation, the resulting plasma cells synthesize and secrete large amounts of the secretory form of IgM (sIgM). Surprisingly, B lymphocytes synthesize an excess of secretory mu chain over the expressed membrane mu chain. However, the sIgM is degraded intracellularly, indicating regulation of IgM expression at the post-translational level. In the present report, we show that the assembly, maturation, and degradation of IgM in 38C B lymphocytes are highly accelerated above a certain threshold temperature. Furthermore, the degradation of sIgM is delayed and takes place by the time the maturation of mIgM in the trans-Golgi is almost completed. Neither chloroquine nor monensin has any effect on this degradation, demonstrating a nonlysosomal pre-trans-Golgi process. In addition, the degradation is of endoglycosidase H-sensitive assembled sIgM molecules. We conclude that the degradation of sIgM in 38C B lymphocytes is a postendoplasmic reticulum, pre-trans-Golgi process. We suggest that this degradation process plays a role in the post-translational regulation of expression of soluble lumenal sIgM.     

Tryptic digestion of human GPIIIa. Isolation and biochemical characterization of the 23 kDa N-terminal glycopeptide carrying the antigenic determinant for a monoclonal antibody (P37) which inhibits platelet aggregation.

Early digestion of pure human platelet glycoprotein IIIa (GPIIIa) leads to a single cleavage of the molecule at 23 kDa far from one of the terminal amino acids. Automated Edman degradation demonstrates that GPIIIa and the smaller (23 kDa) tryptic fragment share the same N-terminal amino acid sequence. A further cleavage occurs in the larger fragment (80 kDa), reducing its apparent molecular mass by 10 kDa. The 23 kDa fragment remains attached to the larger ones in unreduced samples. Stepwise reduction of early digested GPIIIa with dithioerythritol selectively reduces the single disulphide bond joining the smaller (23 kDa) to the larger (80/70 kDa) fragments. Two fractions were obtained by size-exclusion chromatography of early digested GPIIIa after partial or full reduction and alkylation. The larger-size fraction contains the 80/70 kDa fragments, while the 23 kDa fragment is isolated in the smaller. The amino acid compositions of these fractions do not differ very significantly from the composition of GPIIIa; however the 23 kDa fragment contains only 10.2% by weight of sugars and is richer in neuraminic acid. Disulphide bonds are distributed four in the 23 kDa glycopeptide and 20-21 in the 80/70 kDa glycopeptide. The epitope for P37, a monoclonal antibody which inhibits platelet aggregation [Melero & González-Rodríguez (1984) Eur. J. Biochem. 141, 421-427] is situated within the first 17 kDa of the N-terminal region of GPIIIa, which gives a special functional interest to this extracellular region of GPIIIa. On the other hand, the epitopes for GPIIIa-specific monoclonal antibodies, P6, P35, P40 and P97, which do not interfere with platelet aggregation, are located within the larger tryptic fragment (80/70 kDa). Thus, the antigenic areas available in the extracellular surface of GPIIIa for these five monoclonal antibodies are now more precisely delineated.     

OsTDL1A binds to the LRR domain of rice receptor kinase MSP1, and is required to limit sporocyte numbers

Hybrids lose heterotic yield advantage when multiplied sexually via meiosis. A potential alternative breeding system for hybrids is apospory, where female gametes develop without meiosis. Common among grasses, apospory begins in the nucellus, where aposporous initials (AIs) appear near the sexual megaspore mother cell (MeMC). The cellular origin of AIs is obscure, but one possibility, suggested by the mac1 and msp1 mutants of maize and rice, is that AIs are apomeiotic derivatives of the additional MeMCs that appear when genetic control over sporocyte numbers is relaxed. MULTIPLE SPOROCYTES1 (MSP1) encodes a leucine-rich-repeat receptor kinase, which is orthologous to EXS/EMS1 in Arabidopsis. Like mac1 and msp1, exs/ems1 mutants produce extra sporocytes in the anther instead of a tapetum, causing male sterility. This phenotype is copied in mutants of TAPETUM DETERMINANT1 (TPD1), which encodes a small protein hypothesized to be an extracellular ligand of EXS/EMS1. Here we show that rice contains two TPD1-like genes, OsTDL1A and OsTDL1B. Both are co-expressed with MSP1 in anthers during meiosis, but only OsTDL1A and MSP1 are co-expressed in the ovule. OsTDL1A binds to the leucine-rich-repeat domain of MSP1 in yeast two-hybrid assays and bimolecular fluorescence complementation in onion cells; OsTDL1B lacks this capacity. When driven by the maize Ubiquitin1 promoter, RNA interference against OsTDL1A phenocopies msp1 in the ovule but not in the anther. Thus, RNAi produces multiple MeMCs without causing male sterility. We conclude that OsTDL1A binds MSP1 in order to limit sporocyte numbers. OsTDL1A-RNAi lines may be suitable starting points for achieving synthetic apospory in rice.     

Definition of a domain of GLVR1 which is necessary for infection by gibbon ape leukemia virus and which is highly polymorphic between species.

Expression of human GLVR1 in mouse cells confers susceptibility to infection by gibbon ape leukemia virus (GALV), while the normally expressed mouse Glvr-1 does not. Since human and murine GLVR1 proteins differ at 64 positions in their sequences, some of the residues differing between the two proteins are critical for infection. To identify these, a series of hybrids and in vitro-constructed mutants were tested for the ability to confer susceptibility to infection. The results indicated that human GLVR1 residues 550 to 551, located in a cluster of seven of the sites that differ between the human and mouse proteins, are the only residues differing between the two which must be in the human protein form to allow infection. Sequencing of a portion of GLVR1 from the rat (which is infectible) confirmed the importance of this cluster in that it contained the only notable differences between the rat and mouse proteins. This region, which also differs substantially between the rat and the human proteins, therefore exhibits a pronounced tendency for polymorphism.     

Phospholipase A(2) digestion of cardiolipin bound to bovine cytochrome c oxidase alters both activity and quaternary structure.

Phospholipase A(2) from Crotalus atrox hydrolyzes all of the phospholipids that are associated with purified, detergent-solubilized cytochrome c oxidase; less than 0.05 mol cardiolipin (CL)(1) remains bound per mol enzyme. Coincident with the hydrolysis of cardiolipin is a reversible decrease of 45-50% in the electron transport activity of the dodecylmaltoside-solubilized enzyme. Full activity is recoverable (90-98%) by addition of exogenous cardiolipin, but not by either phosphatidylcholine or phosphatidylethanolamine. Unexpectedly, cleavage of cardiolipin causes the dissociation of both subunits VIa and VIb from the enzyme. These are the two subunits that form the major protein-protein contacts between the two monomeric units within the dimeric complex. Although hydrolysis of CL by phospholipase A(2) and loss of these subunits is linked, the reverse process does not occur, i.e., removal of subunits VIa and VIb does not cause dissociation of the two functionally important, tightly bound cardiolipins. Nor does addition of exogenous cardiolipin result in reassociation of the two subunits with the remainder of the complex. We conclude that cardiolipin is not only essential for full electron transport activity, but also has an important structural role in stabilizing the association of subunits VIa and VIb within the remainder of the bovine heart enzyme.     

The Ig1/2 domain of MuSK binds to muscle surface and is involved in acetylcholine receptor clustering

The neuromuscular junction, the synapse between motor neurons and muscle cells, serves as an excellent model for studying synapse formation. Agrin is believed to be released by motor neurons to induce postsynaptic differentiation at the neuromuscular junction. MuSK, a receptor tyrosine kinase, appears to be a key component of the agrin receptor complex. However, how agrin activates MuSK remains unclear. To address this question, we characterized the binding of the MuSK extracellular region to the muscle cell surface. The MuSK ectodomain was found to bind to muscle cells in a manner dependent on stimulation with neural agrin. Moreover, the binding was myotube specific and appeared to be mediated by two regions in the MuSK: one region containing the first and second immunoglobin domains and the other containing the cysteine-rich domain. Importantly, recombinant proteins containing the binding activity can block full-length MuSK binding to muscle cells and agrin-induced AChR clustering. These results suggest that the Ig1/2 domain of MuSK is involved in AChR clustering by binding to the muscle surface.     

Sec1p binds to SNARE complexes and concentrates at sites of secretion.

Proteins of the Sec1 family have been shown to interact with target-membrane t-SNAREs that are homologous to the neuronal protein syntaxin. We demonstrate that yeast Sec1p coprecipitates not only the syntaxin homologue Ssop, but also the other two exocytic SNAREs (Sec9p and Sncp) in amounts and in proportions characteristic of SNARE complexes in yeast lysates. The interaction between Sec1p and Ssop is limited by the abundance of SNARE complexes present in sec mutants that are defective in either SNARE complex assembly or disassembly. Furthermore, the localization of green fluorescent protein (GFP)-tagged Sec1p coincides with sites of vesicle docking and fusion where SNARE complexes are believed to assemble and function. The proposal that SNARE complexes act as receptors for Sec1p is supported by the mislocalization of GFP-Sec1p in a mutant defective for SNARE complex assembly and by the robust localization of GFP-Sec1p in a mutant that fails to disassemble SNARE complexes. The results presented here place yeast Sec1p at the core of the exocytic fusion machinery, bound to SNARE complexes and localized to sites of secretion.     

The head domain of plakophilin-1 binds to desmoplakin and enhances its recruitment to desmosomes. Implications for cutaneous disease.

The contribution of desmosomes to epidermal integrity is evident in the inherited blistering disorder associated with the absence of a functional gene for plakophilin-1. To define the function of plakophilin-1 in desmosome assembly, interactions among the desmosomal cadherins, desmoplakin, and the armadillo family members plakoglobin and plakophilin-1 were examined. In transient expression assays, plakophilin-1 formed complexes with a desmoplakin amino-terminal domain and enhanced its recruitment to cell-cell borders; this recruitment was not dependent on the equimolar expression of desmosomal cadherins. In contrast to desmoplakin-plakoglobin interactions, the interaction between desmoplakin and plakophilin-1 was not mediated by the armadillo repeat domain of plakophilin-1 but by the non-armadillo head domain, as assessed by yeast two-hybrid and recruitment assays. We propose a model whereby plakoglobin serves as a linker between the cadherins and desmoplakin, whereas plakophilin-1 enhances lateral interactions between desmoplakin molecules. This model suggests that epidermal lesions in patients lacking plakophilin-1 are a consequence of the loss of integrity resulting from a decrease in binding sites for desmoplakin and intermediate filaments at desmosomes.     

Pathway of rhinovirus disruption by soluble intercellular adhesion molecule 1 (ICAM-1): an intermediate in which ICAM-1 is bound and RNA is released.

We have examined the pathway of rhinovirus interaction with soluble intercellular adhesion molecule 1 (sICAM-1). Binding of sICAM-1 to rhinovirus serotypes 3 and 14 gives particles with sedimentation coefficients from 145 to 120S, depending on the amount of sICAM-1 bound. The formation of 120S particles is faster and more extensive at a neutral pH than at an acidic pH. A large number of receptors (> 30) can bind to human rhinovirus 3 without disruption. Disruption by sICAM-1 of rhinovirus that yields 80S particles is strongly temperature dependent and is antagonized by a low pH. Interestingly, sICAM-1 remains bound to the viral capsid after RNA is released, although in smaller amounts than those observed for the native virus. We have found heterogeneity both between and within 80S particle preparations in the VP4 content and number of bound receptors. The ability of the virus to remain bound to its receptor during the uncoating process may facilitate the transport of the viral genome into the cytoplasm in vivo.     

Isolation of a nuclear ribonucleoprotein network that contains heterogeneous RNA and is bound to the nuclear envelope.

Rapidly labeled polydispersed nuclear RNA is part of a ribonucleoprotein (RNP) network which in turn is tightly bound to the nuclear membrane. The membranous attachment, therefore, established a connection between chromatin and cytoplasm. The ultrastructure of the RNP network comprises fibrils and granules similar to those observed in intact nuclei. When bound to the nuclear membrane it has the composition of 63% protein, 14% RNA, 0.4% DNA, and 22.6% lipids. The proportion of lipids diminishes to 2.2% when nuclear membrane is not present. Chromatin, nucleoli, and ribosomes are minor contaminants since histones and ribosomal proteins are not detectable in polyacrylamide gel electrophoresis. Nuclear disruption at high pressure in a French pressure cell causes fragmentation of the RNP network into a series of polydispersed RNP particles. Fragmentation can be prevented by using mild pressure, or by disrupting nuclei with high salt buffer and digesting the dispersed chromatin with deoxyribonuclease. A RNP network, almost free of membrane, is also obtained if the nucleus is deprived of its envelope by treatment with Triton X-100. Since no polydispersed RNP particles are found following dissolution of the nuclear membrane, it is assumed that the particles are components of the RNP network whose fragmentation occurs as a consequence of two processes: (a) activation of nuclear nucleases and (b) shearing forces.     

The closed form of Mad2 is bound to Mad1 and Cdc20 at unattached kinetochores

The spindle assembly checkpoint (SAC) ensures accurate chromosome segregation by delaying anaphase onset in response to unattached kinetochores. Anaphase is delayed by the generation of the mitotic checkpoint complex (MCC) composed of the checkpoint proteins Mad2 and BubR1/Bub3 bound to the protein Cdc20. Current models assume that MCC production is catalyzed at unattached kinetochores and that the Mad1/Mad2 complex is instrumental in the conversion of Mad2 from an open form (O-Mad2) to a closed form (C-Mad2) that can bind to Cdc20. Importantly the levels of Mad2 at kinetochores correlate with SAC activity but whether C-Mad2 at kinetochores exclusively represents its complex with Mad1 is not fully established. Here we use a recently established C-Mad2 specific monoclonal antibody to show that Cdc20 and C-Mad2 levels correlate at kinetochores and that depletion of Cdc20 reduces Mad2 but not Mad1 kinetochore levels. Importantly reintroducing wild type Cdc20 but not Cdc20 R132A, a mutant form that cannot bind Mad2, restores Mad2 levels. In agreement with this live cell imaging of fluorescent tagged Mad2 reveals that Cdc20 depletion strongly reduces Mad2 localization to kinetochores. These results support the presence of Mad2-Cdc20 complexes at kinetochores in agreement with current models of the SAC but also argue that Mad2 levels at kinetochores cannot be used as a direct readout of Mad1 levels.