Plasma cell immunoglobulin secretion: arrest is accompanied by alterations of the golgi complex.

Conditions influencing Ig secretion by plasma cells have been studied with suspensions of murine plasma cells and myeloma cells by determining the release of (3)H-Ig after a pulse of biosynthetic labeling with L- [4,5-(3)H]-leucine. Ig secretion is insensitive to a variety of hormones, mediators, cyclic nucleotide derivatives, extracellular calcium depletion, and agents acting on mierotubules or microfilaments; i.e., to a number of factors which are involved in the regulation of secretion by cells with a storage compartment. On the other hand, Ig secretion is markedly inhibited by conditions which (a) lower intracellular calcium levels (ionophore A 23187 in Ca(++)-free medium), (b) induce partial sodium/potassium equilibration (the ionophores monensin and nigericin and, in the case of myeloma cells, ouabain and incubation in K(+)-free medium) or (c) uncouple oxidative phosphorylation. The first two situations are accompanied by striking alterations of the ultrastructural appearance of the Golgi complex, different in each case. These ultrastructural observations, together with autoradiographic experiments after a short pulse with L-[4,5-(3)H]-leucine, have led to the following hypothesis: (a) under Ca(++) depletion (3)H-Ig passes to Golgi vesicles but these vesicles are incapable of fusion or migration and therefore accumulate in exaggerated numbers in the Golgi area; (b) under partial Na(+)/K(+) equilibration, (3)H-Ig passes to Golgi vesicles which have an exaggerated tendency to fuse with other Golgi elements, thereby generating large vacuoles which store increasing amounts of Ig; (c) under energy block, multiple membrane fission and fusion events are inhibited and there is therefore, little intracellular transport of (3)H-Ig or alteration of cell ultrastructure.

The nuclear GTPase cycle: promoting peripheralization?

Numerous Ras-like GTPases function as molecular switches in the cytoplasm, but only one has been identified in the nucleus. This nuclear GTPase and its homologues are known in both yeasts and higher organisms and in all cases they are regulated by guanine-nucleotide-exchange factors. The 'nuclear GTPase cycle' created by these components is implicated in mRNA transport from and protein import to the nucleus, as well as in DNA replication, RNA processing and the regulation of the cell cycle. In this article, Alan Tartakoff and Roger Schneiter propose that this GTPase cycle regulates dispersive functions in the nucleoplasm, an idea that explains many of the observed effects of disrupting the cycle.

Plasma cell immunoglobulin M molecules. Their biosynthesis, assembly, and intracellular transport.

Immunoglobulin M (IgM)-secreting murine plasmablasts have been used to explore the cytologic site(s) of the successive modifications of the polypeptide H and L chains (steps of glycosylation, chain assembly, and polymerization) which occur during intracellular transport (ICT) and the interrelationships between these events. A combination of pulse-chase biosynthetic labeling protocols (using amino acids and sugars), subcellular fractionation, and electron microscope autoradiography was used in conjunction with inhibitors of glycosylation and agents (carboxyl cyanide m-chlorophenyl hydrazone [CCCP] and monensin) which block Ig exit from the rough endoplasmic reticulum (RER) or Golgi cisternae. The data are consistent with the following conclusions: (1) Sugar addition and modification occur in three main steps: (a) en bloc addition of core sugars to nascent H chains, (b) partial trimming of these oligosaccharide chains in the RER, (c) quasiconcerted addition of terminal sugars (galactose, fucose, and sialic acid) in a very distal compartment between monensin-sensitive Golgi cisternae and the cell surface. (2) H and L chain assembly occurs between nascent H chains and a pool of free light chains present in the RER, followed by interchain disulfide bonding and rapid assembly of monomers into J chain-containing pentamers in the RER. Small amounts of various apparently non-obligatory intermediates in polymerization are also formed. (3) Carbohydrate addition is not required for chain assembly, polymerization, and secretion since completely unglycosylated chains (synthesized in the presence of deoxyglucose or tunicamycin) undergo polymerization and are secreted (although at a reduced rate). (4) Surface 8s IgM molecules do not represent a step in the IgM secretory pathway.

Lectin-binding sites as markers of Golgi subcompartments: proximal-to-distal maturation of oligosaccharides.

We investigated the subcellular sites of glycoprotein oligosaccharide maturation by using lectin conjugates to stain lightly-fixed, saponin-permeabilized myeloma cells. At the electron microscopic level, concanavalin A-peroxidase stains the cisternal space of the nuclear envelope, the rough endoplasmic reticulum, and cisternae along the proximal face of the Golgi stack. Conversely, wheat germ agglutinin-peroxidase stains cisternae along the distal face of the Golgi stack, associated vesicles, and the cell surface. These observations confirm the existence of two qualitatively distinct Golgi subcompartments, show that the lectin conjugates can be employed as relatively proximal or distal Golgi markers under conditions of excellent ultrastructural preservation, suggest that the asymmetric distribution of qualitatively distinct oligosaccharides is a property of underlying cellular components and not simply of the principal secretory product, and suggest that the oligosaccharide structure recognized by wheat germ agglutinin is attained during transport from the proximal toward the distal face of the Golgi stack.

Comparative studies of intracellular transport of secretory proteins.

The physiology of protein intracellular transport and secretion by cell types thought to be free from short-term control has been compared with that of the pancreatic acinar cell, using pulse-chase protocols to follow biosynthetically-labeled secretory products. Data previously obtained (Tartakoff, A.M., and P. Vassalli. J. Exp. Med. 146:1332-1345) has shown that plasma-cell immunoglobulin (Ig) secretion is inhibited by respiratory inhibitors, by partial Na/K equilibration effected by the carboxylic ionophore monensin, and by calcium withdrawal effected by the carboxylic ionophore A 23187 in the presence of ethylene glycol bis (beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) and absence of calcium. We report here that both inhibition of respiration and treatment with monensin slow secretion by fibroblasts, and also macrophages and slow intracellular transport (though not discharge per se) by the exocrine pancreatic cells. Attempted calcium withdrawal is inhibitory for fibroblasts but not for macrophages. The elimination of extracellular calcium or addition of 50 mM KCl has no major effect on secretory rate of either fibroblasts or macrophages. Electron microscopic examination of all cell types shows that monensin causes a rapid and impressive dilation of Golgi elements. Combined cell fractionation and autoradiographic studies of the pancreas show that the effect of monensin is exerted at the point of the exit of secretory protein from the Golgi apparatus. Other steps in intracellular transport proceed at normal rates. These observations suggest a common effect of the cytoplasmic Na/K balance at the Golgi level and lead to a model of intracellular transport in which secretory product obligatorily passes through Golgi elements (cisternae?) that are sensitive to monensin. Thus, intracellular transport follows a similar course in both regulated and nonregulated secretory cells up to the level of distal Golgi elements.

The response of the Golgi complex to microtubule alterations: the roles of metabolic energy and membrane traffic in Golgi complex organization.

A striking example of the interrelation between the Golgi complex (GC) and microtubules is the reversible fragmentation and dispersal of the GC which occurs upon microtubule depolymerization. We have characterized dispersal of the GC after nocodazole treatment as well as its recovery from the dispersed state by immunofluorescent localization of beta 1, 4-galactosyltransferase in Madin-Darby bovine kidney cells. Immunofluorescent anti-tubulin staining allowed simultaneous examination of the microtubule array. Based on our results, dispersal can be divided into a three-step process: microtubule depolymerization, GC fragmentation, and fragment dispersal. In cells treated with metabolic inhibitors after microtubule depolymerization, neither fragmentation nor dispersal occur, despite the absence of assembled microtubules. Thus, fragmentation is energy dependent and not tightly linked to microtubule depolymerization. The slowing of fragmentation and dispersal by monensin or ammonium chloride, as well as progressive inhibition at less than 34 degrees C, suggest that ongoing membrane traffic is required for these processes. Similarly, recovery may be separated into four steps: microtubule depolymerization, GC fragment centralization, fragment coalescence, and polarization of the reticular GC network. Fragment centralization and coalescence were arrested by metabolic inhibitors, despite the presence of microtubules. Neither monensin nor ammonium choride inhibited GC recovery. Partial inhibition of recovery at reduced temperatures paralleled the extent of microtubule assembly. These data demonstrate that dispersal and recovery are multi-step operations, and that the individual steps differ in temperature dependence, energy dependence, and sensitivity to ionic perturbation. GC distribution and microtubule status have also been clearly dissociate, thereby proving that organization of the GC is an active process that is not simply determined by microtubule binding. Furthermore, the results indicate that ongoing intra-GC membrane traffic may participate in fragmentation and dispersal.

Characterization of cytoplasmically oriented Golgi proteins with a monoclonal antibody.

BALB/c mice were repeatedly immunized with a galactosyl transferase-rich microsomal fraction of rat myeloma cells. Spleen cells were subsequently fused with Sp2/0 mouse myeloma cells, the resulting hybridomas were cloned, and their secreted Ig was screened for reactivity with antigens belonging to the Golgi complex. One such monoclonal antibody, 6F4C5, gave especially intense immunofluorescent staining of the Golgi area of myeloma cells and fibroblasts. It recognized two proteins bands on immunoblots of gel-fractionated cell lysates: a major one with an estimated Mr of 54,000 and a minor one at 86,000. Both proteins were concentrated in microsomal fractions isolated at low ionic strength. They were hydrophilic judging from partitioning of a Triton X-114 cell lysate. Both were cytoplasmically oriented as demonstrated by protease and high KCl treatments of postmitochondrial supernatants and microsomal fractions. Neither was retained by columns of insolubilized wheat germ agglutinin or concanavalin A, which suggests that they are not glycoproteins. Their more detailed location in the Golgi complex was studied by immunoelectron microscopy, using a saponin permeabilization procedure and peroxidase-conjugated reagents. The observed staining was restricted to two or three cisternae in the medial part of the stack. Nevertheless, differential centrifugation experiments indicated that the two antigens may be recovered in distinct subcellular fractions: this may be related to the unexpected observation that rather low salt concentrations strip the antigens from microsomal fraction.

Cell fractionation studies on the guinea pig pancreas. Redistribution of exocrine proteins during tissue homogenization.

A double-label protocol was used to estimate the extent of leakage and relocation artifacts that affect exocrine pancreatic proteins in cell fractionation experiments. Guinea pig pancreatic lobules were pulsed in vitro with a mixture of 14C-amino acids to enable the lobules to produce and process endogenously labeled exocrine proteins. At the end of the pulse (10 min) or after an appropriate chase interval, the lobules were homogenized in 0.3 M sucrose to which a complete mixture of 3H-labeled exocrine pancreatic proteins was added as an exogenous tracer. The distribution of both labels was studied in each cell fraction of interest at the level of TCA-insoluble proteins and individual exocrine proteins resolved by using a two-dimensional gel system. Based on the premises that the exogenous and endogenous label behave identically during homogenization-fractionation and that all endogenously labeled exocrine proteins found in the postmicrosomal supernate come from intracellular compartments ruptured during tissue homogenization, a series of equations was derived to quantitate leakage and adsorption and to define the ratio of endogenous label still in its primary location to total label (primary location index or PLI) for each cell fraction. Leakage was found to be uniform for all exocrine proteins, but unequal in extent from different cell compartments (condensing vacuoles is greater than zymogen granules is greater than rough endoplasmic reticulum) ; it increased with exposure to shearing forces especially in the case of zymogen granules and condensing vacuoles, and was substantially reduced from rough microsomes by adding 10 mM KCl to the homogenization media. Relocation of exogenous label by adsorption to other subcellular components was extensive (approximately 55%), uneven (free polysomes is greater than rough microsomes is greater than smooth microsomes and zymogen granules), preferential (cationic proteins are massively adsorbed to ribosomes and membranes, resulting in a complementary enrichment of the post-microsomal supernate with anionic exocrine proteins), and reversible (with successive 50-100 mM KCl washes). After correction for adsorption and leakage, the kinetics of intracellular transport derived from cell fractionation data were found to be nearly identical to those obtained from quantitative autoradiographic studies.

Dynamics and longevity of the glycolipid-anchored membrane protein, Thy-1.

Thy-1 and a number of other proteins are anchored to the outer hemi-leaflet of membranes by a glycolipid moiety containing ethanolamine phosphate, mannose, glucosamine, and phosphatidylinositol. They nevertheless have the striking property of being able to transduce signals across the plasma membrane. We here demonstrate, for the BW5147 murine T lymphoma, that (a) greater than 90% of Thy-1 is at the cell surface, (b) Thy-1 is about one order of magnitude less concentrated in coated pits than the transferrin receptor or H-2 antigens, (c) Thy-1 undergoes at most very limited endocytosis or diacytosis, and (d) Thy-1 has an unusually slow turnover rate. Several similar observations have also been made for a second glycolipid-anchored protein, the T cell activating protein. Thus, the absence of cytoplasmic and trans-membrane domains may result in lipid-anchored proteins being confined to the cell surface and being free from constraints which affect the turnover of transmembrane proteins.

Isolation and characterization of Saccharomyces cerevisiae mRNA transport-defective (mtr) mutants.

To understand the mechanisms of mRNA transport in eukaryotes, we have isolated Saccharomyces cerevisiae temperature-sensitive (ts) mutants which accumulate poly(A)+ RNA in the nucleus at the restrictive temperature. A total of 21 recessive mutants were isolated and classified into 16 complementation groups. Backcrossed mRNA transport-defective strains from each complementation group have been analyzed. A strain which is ts for heat shock transcription factor was also analyzed since it also shows nuclear accumulation of poly(A)+ RNA at 37 degrees C. At 37 degrees C the mRNA of each mutant is characterized by atypically long polyA tails. Unlike ts pre-mRNA splicing mutants, these strains do not interrupt splicing of pre-mRNA at 37 degrees C; however four strains accumulate oversized RNA polymerase II transcripts. Some show inhibition of rRNA processing and a further subset of these strains is also characterized by inhibition of tRNA maturation. Several strains accumulate nuclear proteins in the cytoplasm when incubated at semipermissive temperature. Remarkably, many strains exhibit nucleolar fragmentation or enlargement at the restrictive temperature. Most strains show dramatic ultrastructural alterations of the nucleoplasm or nuclear membrane. Distinct mutants accumulate poly(A)+ RNA in characteristic patterns in the nucleus.

Metabolic correction of defects in the lipid anchoring of Thy-1 in lymphoma mutants.

Many plasma membrane proteins, including Thy-1, are anchored by a carboxyl terminal glycophospholipid. This unit is absent from the Thy-1 of several lymphoma mutants that synthesize the Thy-1 polypeptide but fail to express it at the cell surface. Recessive mutants of complementation groups A to C, E, and F contain Thy-1 mRNA of normal size, which suggests that their Thy-1 polypeptide is normal. To identify possible metabolic lesions, each mutant was grown with various supplements. The class F and B mutants exhibited a reversible induction of surface lipid anchored Thy-1 when grown with the aminoglycoside G418. Other aminoglycosides, sugars, and ethanolamine were inactive. These unexpected observations are discussed in the context of lipid anchor biosynthesis.

How to make a glycoinositol phospholipid anchor.

Essentially all eukaryotic cells express proteins on their surface that are anchored by a glycoinositol phospholipid. This anchor moiety may endow such proteins with unusual properties. The definition of the biosynthetic path that constructs these anchors is now in its final stages. Mutations that interrupt this path are, remarkably, compatible with survival of cells in culture, but are associated with at least one human disease.

Internalization of type 1 complement receptors and de novo multivesicular body formation during chemoattractant-induced endocytosis in human neutrophils.

Upon activation of human neutrophils by chemoattractants, functionally important proteins are rapidly transported from intracellular granules and storage vesicles to the plasma membrane. This is accompanied by a marked increase in the rate of endocytosis and by ligand-independent internalization of type 1 complement receptors (CR1). To define the pathway of endocytosis, we used gold-conjugated BSA in a pulse-chase protocol. This tracer was initially internalized into small endocytic vesicles which rapidly traversed the cytoplasm and coalesced to form large, conspicuous multivesicular bodies. Within 5 min after addition of the chemoattractant, multivesicular bodies contained > 60% of the cell-associated BSA-gold. CR1 colocalized with the endocytic tracer in both the early endosomes and multivesicular bodies. In unstimulated cells, there was much less uptake of BSA-gold and multivesicular bodies were rarely seen. Using the acidotropic amine, DAMP, and anti-DNP antibodies, we found that the multivesicular bodies were acidified but the early endosomes did not concentrate DAMP. Neither the early endosomes nor the multivesicular bodies initially contained the lysosomal membrane antigens hLAMP 1 or 2, but hLAMP-positive structures subsequently joined the multivesicular bodies. The rapid activation of the endocytic pathway upon stimulation of neutrophils allowed us to visualize the de novo formation and maturation of multivesicular bodies. Our observations suggest that vesicles containing ion pumps and acid hydrolases fuse with multivesicular bodies, giving them characteristics of lysosomes, and that these are the probable sites of degradation of CR1. The observations do not support models which would require transport of CR1 from multivesicular bodies to defined, pre-existing lysosomes for degradation.

Two different mutants blocked in synthesis of dolichol-phosphoryl-mannose do not add glycophospholipid anchors to membrane proteins: quantitative correction of the phenotype of a CHO cell mutant with tunicamycin.

The addition of glycophospholipid (GPL) anchors to certain membrane proteins occurs in the rough endoplasmic reticulum and is essential for transport of the proteins to the plasma membrane. Limited circumstantial evidence suggests that dolichol-phosphoryl-mannose (DPM) is a donor of mannose residues of these anchors. We here report studies of a CHO cell mutant (B421) transfected to express the GPL-anchored protein, placental alkaline phosphatase (AP). Only a few transfectants were found to express GPL-anchored AP on their surface, and these clones synthesized DPM. Moreover, and most strikingly, when surface AP-negative transfectants were treated with tunicamycin to cause accumulation of DPM, these cells expressed lipid-anchored AP. Fusion of a cloned surface AP-negative transfectant of B421 with the Thy-1-class E mutant thymoma, which is also deficient in DPM synthesis, produced hybrids that synthesized DPM and expressed AP and Thy-1. Thus, two mutations can interrupt DPM synthesis, and three sets of observations point to an essential role of DPM for addition of GPL anchors.

Anchoring and degradation of glycolipid-anchored membrane proteins by L929 versus by LM-TK- mouse fibroblasts: implications for anchor biosynthesis.

Although many cells anchor surface proteins via moieties that are sensitive to phosphatidylinositol-specific phospholipase C (PI-PLC), the anchor moieties of surface proteins of mouse L929 cells resist PI-PLC. By constructing stable hybrids between L929 and lymphoma cells that express glycolipid-anchored proteins in a PI-PLC-sensitive form, we show that PI-PLC resistance behaves as a recessive trait. Since putative mannolipid precursors of the lipid anchors bear alkali-labile substituents which make them resist PI-PLC, these observations are most simply interpreted by postulating that L929 lacks a critical anchor deacylase. Unlike the L929 cell line, two of its descendants, the LM cell line and its thymidine kinase-negative variant (LM-TK-), do not express glycolipid-anchored proteins on their surface. Moreover, unlike L929 cells, LM-TK- cells rapidly inactivate at least one lipid-anchored enzyme in a compartment sensitive to acidotropic amines and leupeptin. By fusion of LM-TK- cells to mouse Thy-1- lymphoma mutants and monitoring of surface expression of lipid-anchored proteins, we assign LM-TK- to lymphoma mutant complementation group H. This genetic assignment is matched by analysis of mannolipids of L929, LM-TK-, wild-type, and class H lymphoma mutant cells: striking similarities are seen between the two wild-type cells by contrast to the mutants. Since the differences pertain to lipids which have properties consistent with their being anchor precursors, we suggest that LM-TK- has a lesion in the synthesis of anchor precursor mannolipids.

A DEAD-box-family protein is required for nucleocytoplasmic transport of yeast mRNA.

An enormous variety of primary and secondary mRNA structures are compatible with export from the nucleus to the cytoplasm. Therefore, there seems to be a mechanism for RNA export which is independent of sequence recognition. There nevertheless is likely to be some relatively uniform mechanism which allows transcripts to be packaged as ribonucleoprotein particles, to gain access to the periphery of the nucleus and ultimately to translocate across nuclear pores. To study these events, we and others have generated temperature-sensitive recessive mRNA transport (mtr) mutants of Saccharomyces cerevisiae which accumulate poly(A)+ RNA in the nucleus at 37 degrees C. Several of the corresponding genes have been cloned. Upon depletion of one of these proteins, Mtr4p, conspicuous amounts of nuclear poly(A)+ RNA accumulate in association with the nucleolus. Corresponding dense material is also seen by electron microscopy. MTR4 is essential for growth and encodes a novel nuclear protein with a size of approximately 120 kDa. Mtr4p shares characteristic motifs with DEAD-box RNA helicases and associates with RNA. It therefore may well affect RNA conformation. It shows extensive homology to a human predicted gene product and the yeast antiviral protein Ski2p. Critical residues of Mtr4p, including the mtr4-1 point mutation, have been identified. Mtr4p may serve as a chaperone which translocates or normalizes the structure of mRNAs in preparation for export.

Atypical mannolipids characterize Thy-1-negative lymphoma mutants.

Essentially all eukaryotic cells, including murine lymphomas, express surface proteins, such as Thy-1, which are anchored by a phosphoinositol mannolipid. Putative mannolipid anchor precursors can be detected in these cells. Six distinct Thy-1-negative lymphoma mutants lack complete mannolipids, and three mutants synthesize atypical mannolipids. The absence of complete mannolipids can account for the lack of expression of multiple mannolipid-anchored proteins and may also account for the lack of lipid anchoring in the human disease paroxysmal nocturnal hemoglobinuria. Structural information on the mannolipids of wild-type and mutant cells indicates that anchor biosynthesis in these cells may involve both transmembrane flip-flop of intermediates and a deacylation step.

Addition of lipid substituents of mammalian protein glycosylphosphoinositol anchors.

A single metabolic path leading to synthesis of ether lipids is known in animal cells, the major products of which are plasmalogens. To learn whether this peroxisomal path is also responsible for the synthesis of base-resistant lipid components of glycosylphosphoinositol (GPI)-anchored membrane proteins, we have investigated the structure of anchor precursor mannolipids both in wild-type cells (CHO-K1 and a macrophage-like line, RAW 264.7) and in two corresponding mutant cells in which ether lipid biosynthesis is severely impaired. We observe that the precursor mannolipids of both the wild-type and mutant cells do not include alkylglycerol. Nevertheless, both wild-type and mutant cells express cell surface GPI-anchored placental alkaline phosphatase (AP) which includes alkali-resistant hydrophobic chains in its anchor moiety. Thus, (i) in normal AP GPI anchor synthesis, any ether-linked substituents must be added either immediately before, during, or after anchor addition to AP, and (ii) the classical peroxisomal path for ether lipid synthesis appears not to contribute to the synthesis of GPI anchors.

A yeast acetyl coenzyme A carboxylase mutant links very-long-chain fatty acid synthesis to the structure and function of the nuclear membrane-pore complex.

The conditional mRNA transport mutant of Saccharomyces cerevisiae, acc1-7-1 (mtr7-1), displays a unique alteration of the nuclear envelope. Unlike nucleoporin mutants and other RNA transport mutants, the intermembrane space expands, protuberances extend from the inner membrane into the intermembrane space, and vesicles accumulate in the intermembrane space. MTR7 is the same gene as ACC1, encoding acetyl coenzyme A (CoA) carboxylase (Acc1p), the rate-limiting enzyme of de novo fatty acid synthesis. Genetic and biochemical analyses of fatty acid synthesis mutants and acc1-7-1 indicate that the continued synthesis of malonyl-CoA, the enzymatic product of acetyl-CoA carboxylase, is required for an essential pathway which is independent from de novo synthesis of fatty acids. We provide evidence that synthesis of very-long-chain fatty acids (C26 atoms) is inhibited in acc1-7-1, suggesting that very-long-chain fatty acid synthesis is required to maintain a functional nuclear envelope.

mRNA transport in yeast: time to reinvestigate the functions of the nucleolus.

Nucleocytoplasmic transport of mRNA is vital to gene expression and may prove to be key to its regulation. Genetic approaches in Saccharomyces cerevisiae have led to the identification of conditional mutants defective in mRNA transport. Mutations in approximately two dozen genes result in accumulation of transcripts, trapped at various sites in the nucleus, as detected by in situ hybridization. Phenotypic and molecular analyses of many of these mRNA transport mutants suggest that, in yeast, the function of the nucleus is not limited to the biogenesis of pre-ribosomes but may also be important for transport of poly(A)+ RNA. A similar function of the animal cell nucleolus is suggested by several observations.