Analysis of BHK cell growth kinetics after microinjection of catalytic subunit of cyclic AMP-dependent protein kinase.

The effect of catalytic subunit (C) of cyclic AMP-dependent protein kinase on cell growth kinetics of BHK cells was assessed by microinjection with chicken erythrocyte ghosts as vehicles for introduction of the protein into the cytosol of large populations of cells. The advantage in using chicken erythrocytes for microinjection is that the inactive erythrocyte nuclei serve as a probe for identifying and analyzing microinjection events. By utilizing this procedure, BHK cells were microinjected with an amount of C that was 5- to 10-fold greater than their endogenous levels. Growth kinetics were analyzed by [3H]thymidine incorporation and autoradiography. Cells were stained after autoradiography to more clearly reveal the chicken nuclei, and at each time point, cells were categorized into four groups: (i) not microinjected, not in S phase, (ii) not microinjected, in S phase, (iii) microinjected, not in S phase, (iv) microinjected, in S phase. Those cells not microinjected served as internal controls. Two experimental protocols were used to test the notion that C is involved in blocking cell progression through G1 phase of the cell cycle. First, cells were arrested in G0 phase by serum deprivation, microinjected with C or control proteins, and stimulated to proceed to S phase by the addition of serum or purified growth factors. Second, cells were collected in mitosis, microinjected with C or control proteins, and stimulated to proceed to S phase by the addition of serum. The results of these studies indicate that a 5- to 10-fold increase in the intracellular concentration of C is not a sufficient signal to arrest cell growth in G1 phase. Thus, growth-inhibitory effects of cyclic AMP on BHK cells are unlikely to be the result of activation of cyclic AMP-dependent protein kinase.     

Recycling of Golgi-resident Glycosyltransferases through the ER Reveals a Novel Pathway and Provides an Explanation for Nocodazole-induced Golgi Scattering

During microtubule depolymerization, the central, juxtanuclear Golgi apparatus scatters to multiple peripheral sites. We have tested here whether such scattering is due to a fragmentation process and subsequent outward tracking of Golgi units or if peripheral Golgi elements reform through a novel recycling pathway. To mark the Golgi in HeLa cells, we stably expressed the Golgi stack enzyme N-acetylgalactosaminyltransferase-2 (GalNAc-T2) fused to the green fluorescent protein (GFP) or to an 11–amino acid epitope, VSV-G (VSV), and the trans/TGN enzyme β1,4-galactosyltransferase (GalT) fused to GFP. After nocodazole addition, time-lapse microscopy of GalNAc-T2–GFP and GalT–GFP revealed that scattered Golgi elements appeared abruptly and that no Golgi fragments tracked outward from the compact, juxtanuclear Golgi complex. Once formed, the scattered structures were relatively stable in fluorescence intensity for tens of minutes. During the entire process of dispersal, immunogold labeling for GalNAc-T2–VSV and GalT showed that these were continuously concentrated over stacked Golgi cisternae and tubulovesicular Golgi structures similar to untreated cells, suggesting that polarized Golgi stacks reform rapidly at scattered sites. In fluorescence recovery after photobleaching over a narrow (FRAP) or wide area (FRAP-W) experiments, peripheral Golgi stacks continuously exchanged resident proteins with each other through what appeared to be an ER intermediate. That Golgi enzymes cycle through the ER was confirmed by microinjecting the dominant-negative mutant of Sar1 (Sar1p^dn) blocking ER export. Sar1p^dn was either microinjected into untreated or nocodazole-treated cells in the presence of protein synthesis inhibitors. In both cases, this caused a gradual accumulation of GalNAc-T2–VSV in the ER. Few to no peripheral Golgi elements were seen in the nocodazole-treated cells microinjected with Sar1p^dn. In conclusion, we have shown that Golgi-resident glycosylation enzymes recycle through the ER and that this novel pathway is the likely explanation for the nocodazole-induced Golgi scattering observed in interphase cells.     

Immunoisolation and Characterization of a Subdomain of the Endoplasmic Reticulum That Concentrates Proteins Involved in COPII Vesicle Biogenesis

Rubella virus E1 glycoprotein normally complexes with E2 in the endoplasmic reticulum (ER) to form a heterodimer that is transported to and retained in the Golgi complex. In a previous study, we showed that in the absence of E2, unassembled E1 subunits accumulate in a tubular pre-Golgi compartment whose morphology and biochemical properties are distinct from both rough ER and Golgi. We hypothesized that this compartment corresponds to hypertrophied ER exit sites that have expanded in response to overexpression of E1. In the present study we constructed BHK cells stably expressing E1 protein containing a cytoplasmically disposed epitope and isolated the pre-Golgi compartment from these cells by cell fractionation and immunoisolation. Double label indirect immunofluorescence in cells and immunoblotting of immunoisolated tubular networks revealed that proteins involved in formation of ER-derived transport vesicles, namely p58/ERGIC 53, Sec23p, and Sec13p, were concentrated in the E1-containing pre-Golgi compartment. Furthermore, budding structures were evident in these membrane profiles, and a highly abundant but unknown 65-kDa protein was also present. By comparison, marker proteins of the rough ER, Golgi, and COPI vesicles were not enriched in these membranes. These results demonstrate that the composition of the tubular networks corresponds to that expected of ER exit sites. Accordingly, we propose the name SEREC (smooth ER exit compartment) for this structure.     

Scattered Golgi Elements during Microtubule Disruption Are Initially Enriched in Trans-Golgi Proteins

We have addressed the question of whether or not Golgi fragmentation, as exemplified by that occurring during drug-induced microtubule depolymerization, is accompanied by the separation of Golgi subcompartments one from another. Scattering kinetics of Golgi subcompartments during microtubule disassembly and reassembly following reversible nocodazole exposure was inferred from multimarker analysis of protein distribution. Stably expressed α-2,6-sialyltransferase and N-acetylglucosaminyltransferase-I (NAGT-I), both C-terminally tagged with the myc epitope, provided markers for the trans-Golgi/trans-Golgi network (TGN) and medial-Golgi, respectively, in Vero cells. Using immunogold labeling, the chimeric proteins were polarized within the Golgi stack. Total cellular distributions of recombinant proteins were assessed by immunofluorescence (anti-myc monoclonal antibody) with respect to the endogenous protein, β-1,4-galactosyltransferase (GalT, trans-Golgi/TGN, polyclonal antibody). ERGIC-53 served as a marker for the intermediate compartment). In HeLa cells, distribution of endogenous GalT was compared with transfected rat α-mannosidase II (medial-Golgi, polyclonal antibody). After a 1-h nocodazole treatment, Vero α-2,6-sialyltransferase and GalT were found in scattered cytoplasmic patches that increased in number over time. Initially these structures were often negative for NAGT-I, but over a two- to threefold slower time course, NAGT-I colocalized with α-2,6-sialyltransferase and GalT. Scattered Golgi elements were located in proximity to ERGIC-53-positive structures. Similar trans-first scattering kinetics was seen with the HeLa GalT/α-mannosidase II pairing. Following nocodazole removal, all cisternal markers accumulated at the same rate in a juxtanuclear Golgi. Accumulation of cisternal proteins in scattered Golgi elements was not blocked by microinjected GTPγS at a concentration sufficient to inhibit secretory processes. Redistribution of Golgi proteins from endoplasmic reticulum to scattered structures following brefeldin A removal in the presence of nocodazole was not blocked by GTPγS. We conclude that Golgi subcompartments can separate one from the other. We discuss how direct trafficking of Golgi proteins from the TGN/trans-Golgi to endoplasmic reticulum may explain the observed trans-first scattering of Golgi transferases in response to microtubule depolymerization.     

A monoclonal antibody against a 135-K Golgi membrane protein.

A monoclonal antibody ( 53FC3 ) has been produced against a Golgi membrane protein with a mol. wt. of 135 000 which was originally identified using a polyclonal antiserum. Treatment of isolated, intact Golgi vesicles with protease caused a decrease in mol. wt. of 5000-10 000, whereas in the presence of Triton X-100, the protein was completely degraded. This shows that the protein spans the bilayer and that most of its mass is on the luminal side of Golgi membranes. Using two immunoelectron microscopic techniques, the protein was found in one or two cisternae on one side of the Golgi stack which, in normal rat kidney cells, had 4-6 cisternae. As an illustration of the use to which this monoclonal antibody can be put we present a light microscopic study of the disassembly and reassembly of the Golgi complex during mitosis.     

Clostridium chauvoei: Immunological characterization of Antigenic Preparations

Clostridium chauvoei is an anaerobic and sporulated bacterium which produces blackleg in cattle, sheep and other ungulates. Elsewhere we have studied the immunogenic power of the following antigenic preparations in mice: cellular extract (CE), a suspension of flagella (F), formolinized cells (FC) and heated cells (ΘC), and the immunogenic power of the CE in guinea pigs. The aim of this work was to characterize these immunogenic preparations by SDS-PAGE and immunoblotting. When SDS-PAGE was carried out F showed four protein bands of 86, 59, 51 and 47 kDa; CE showed 10 major bands of 86, 75, 62, 55, 47, 41, 30, 28, 25 and 16 kDa; FC six major bands of 86, 75, 62, 53, 46 and 16 kDa; and ΘC 11 bands, the major ones having 80, 41 and 16 kDa. All four preparations share the flagellar protein band of 47 kDa except ΘC. The formol-treatment reduces the number of the protein bands when it is compared with those of CE. The heat-treatment also reduces the number of the bands compared with CE and FC. When immunoblotting is analysed it can be seen that CE induces the antibodies that react with the 47 kDa protein band, the same ones are induced by FC but not by ΘC. On the other hand, CE induces antibodies that react with the 86 kDa protein band, which is believed to be a polymeric form of a flagellin monomer. According to these results and those obtained in the protection tests, we can conclude that: (1) flagella are in part, responsible for the immunogenicity of the strain; (2) the buffer used to obtain the CE extracts flagellar and somatic antigens; (3) the formol-treatment reduces the protein bands compared with CE, but retains their immunogenic power; and (4) the heat-treatment reduces immunogenic power which is seen by its minor protein bands number and by its lower immunoprotective power.     

Direct visualization of protein transport and processing in the living cell by microinjection of specific antibodies

We have prepared polyclonal antibodies to the cytoplasmic portion of the envelope glycoprotein G of vesicular stomatitis virus (VSV) by using synthetic peptides corresponding to either the 22 or 11 ultimate carboxy-terminal residues of the G as immunogens. When antibodies to the 22 residue peptide are microinjected into monolayer baby hamster kidney cells before or shortly after infection with wild-type VSV, G protein accumulates in large intracellular patches and little G is observed in the Golgi complex or at the cell surface. In contrast, when antibodies to the 11 residue peptide are injected, no such patches are observed and G protein is seen colocalized with the injected antibody at the endoplasmic reticulum, in the Golgi complex, in transport vesicles, and at the plasma membrane. Microinjection of these antibodies does not disturb the pathway or kinetics of G-protein transport. In cells infected with a temperature-sensitive mutant of VSV, 045, the glycoprotein accumulates in the endoplasmic reticulum at 39.8 degrees C, but rapidly moves through the Golgi apparatus and then to the cell surface after a temperature shift-down to 32 degrees C. Using rhodamine-coupled antibodies to the 11 residue peptide, a microscope stage equipped for precise temperature control, and a silicon intensifier target video camera, we can visualize by video light microscopy the synchronized exocytotic transport of the G protein directly in the living cell.     

Cell-free transfer of the vesicular stomatitis virus G protein from an endoplasmic reticulum compartment of baby hamster kidney cells to a rat liver Golgi apparatus compartment for Man8-9 to Man5 processing.

We report the reconstitution of the transfer of a membrane glycoprotein (vesicular stomatitis virus glycoprotein, VSV-G protein) from endoplasmic reticulum to Golgi apparatus and its subsequent Man8-9GlcNAc2 to Man5GlcNAc2 processing in a completely cell-free system. The acceptor was Golgi apparatus from rat liver immobilized on nitrocellulose. The endoplasmic reticulum donor was from homogenates of VSV-G-infected BHK cells. Nucleoside triphosphate plus cytosol-dependent transfer and processing of radiolabeled VSV-G protein was observed with donor from BHK cells infected at 37 degrees C with wild-type VSV or at the permissive temperature of 34 degrees C with the ts045 mutant. With Golgi apparatus as acceptor, specific transfer at 37 degrees C in the presence of nucleoside triphosphate was eightfold that at 4 degrees C or in the absence of ATP. About 40% of the VSV-G protein transferred was processed to the Man5GlcNAc2 form. Processing was specific for cis Golgi apparatus fractions purified by preparative free-flow electrophoresis. Fractions derived from the trans Golgi apparatus were inactive in processing. With the ts045 temperature-sensitive mutant, transfer and processing were much reduced even in the complete system when microsomes were from cells infected with mutant virus and incubated at the restrictive temperature of 39.5 degrees C but were able to proceed at the permissive temperature of 34 degrees C. Thus, Man8-9GlcNAc2 to Man5GlcNAc2 processing of VSV-G protein occurs following transfer in a completely cell-free system using immobilized intact Golgi apparatus or cis Golgi apparatus cisternae as the acceptor and shows temperature sensitivity, donor specificity, requirement for ATP, and response to inhibitors similar to those exhibited by transfer and processing of VSV-G protein in vivo.     

Effects of acceptor composition and mechanism of ABCG1-mediated cellular free cholesterol efflux

Among the known mechanisms of reverse cholesterol transport (RCT), ATP binding cassette transporter G1 (ABCG1)-mediated free cholesterol (FC) transport is the most recent and least studied. Here, we have characterized the efficiencies of different acceptors using baby hamster kidney (BHK) cells transfected with human ABCG1 cDNA, which is inducible upon treatment with mifepristone. When normalized on particle number and particle surface area, the acceptor efficiency for FC efflux was as follows: small unilamellar vesicles (SUV)>LDL>reconstituted HDL>HDL(2) = HDL(3). Based on phospholipid content, the order was reversed. ABCG1 also mediated phospholipid efflux to human serum and HDL(3). ABCG1-mediated FC efflux correlated significantly with a number of HDL subfractions and components in serum collected from 25 normolipidemic individuals: apolipoprotein A-II (apoA-II) (r(2) = 0.7), apolipoprotein A-I (apoA-I) (r(2) = 0.5), HDL-C (r(2) = 0.4), HDL-PL (r(2) = 0.4), alpha-2 HDL (r(2) = 0.4), and prebeta HDL (r(2) = 0.2). ABCG1 did not enhance influx of FC or cholesteryl oleyl ether (COE) when cells were incubated with radiolabeled HDL(3). ABCG1 expression did not increase the association of HDL(3) with cells. Compared with control cells, ABCG1 expression significantly increased the FC pool available for efflux and the rate constant for efflux. In conclusion, composition and particle size determine the acceptor efficiency for ABCG1-mediated efflux. ABCG1 increases cell membrane FC pools and changes its rate of desorption into the aqueous phase without enhancing the association with the acceptor.     

Identification of an intermediate compartment involved in protein transport from endoplasmic reticulum to Golgi apparatus

We have studied the role of a previously described tubulovesicular compartment near the cis-Golgi apparatus in endoplasmic reticulum (ER)-to-Golgi protein transport by light and immunoelectron microscopy in Vero cells. The compartment is defined by a 53-kDa transmembrane protein designated p53. When transport of the vesicular stomatitis virus strain ts045 G protein was arrested at 39.5 degrees C, the G protein accumulated in the ER but had access to the p53 compartment. At 15 degrees C, the G protein was exported from the ER into the p53 compartment which formed a compact structure composed of vesicular and tubular profiles in close proximity to the Golgi. Upon raising the temperature to 32 degrees C, the G protein migrated through the Golgi apparatus while the p53 compartment resumed its normal structure again. These results establish the p53 compartment as the 15 degrees C intermediate of the ER-to-Golgi protein transport pathway.     

A Rab1 mutant affecting guanine nucleotide exchange promotes disassembly of the Golgi apparatus

The Golgi apparatus is a dynamic organelle whose structure is sensitive to vesicular traffic and to cell cycle control. We have examined the potential role for rab1a, a GTPase previously associated with ER to Golgi and intra-Golgi transport, in the formation and maintenance of Golgi structure. Bacterially expressed, recombinant rab1a protein was microinjected into rat embryonic fibroblasts, followed by analysis of Golgi morphology by fluorescence and electron microscopy. Three recombinant proteins were tested: wild-type rab, mutant rab1a(S25N), a constitutively GDP-bound form (Nuoffer, C., H. W. Davidson, J. Matteson, J. Meinkoth, and W. E. Balch, 1994. J. Cell Biol. 125: 225- 237), and mutant rab1a(N124I) defective in guanine nucleotide binding. Microinjection of wild-type rab1a protein or a variety of negative controls (injection buffer alone or activated ras protein) did not affect the appearance of the Golgi, as visualized by immunofluorescence of alpha-mannosidase II (Man II), used as a Golgi marker. In contrast, microinjection of the mutant forms promoted the disassembly of the Golgi stacks into dispersed vesicular structures visualized by immunofluorescence. When S25N-injected cells were analyzed by EM after immunoperoxidase labeling, Man II was found in isolated ministacks and large vesicular elements that were often surrounded by numerous smaller unlabeled vesicles resembling carrier vesicles. Golgi disassembly caused by rab1a mutants differs from BFA-induced disruption, since beta- COP remains membrane associated, and Man II does not redistribute to the ER. BFA can still cause these residual Golgi elements to fuse and disperse, albeit at a slower rate. Moreover, BFA recovery is incomplete in the presence of rab1 mutants or GTP gamma S. We conclude that GTP exchange and hydrolysis by GTPases, specifically rab1a, are required to form and maintain normal Golgi stacks. The similarity of Golgi disassembly seen with rab1a mutants to that occurring during mitosis, may point to a molecular basis involving rab1a for fragmentation of the Golgi apparatus during cell division.     

Effects of growth conditions on cyclic nucleotide phosphodiesterases of cultured fibroblasts.

Cyclic nucleotide phosphodiesterase activities of baby hamster kidney cells (BHK) grown in surface cultures were altered by modifying growth conditions. The untransformed BHK cells grown in medium containing 10% fetal calf serum showed non-linear LineweaverBurk plots for cyclic AMP phosphodiesterase activity with apparent Michaelis constants for cyclic AMP of approximately 5 and 30 muM. When these cells were placed in medium containing 1% fetal calf serum, linear kinetic plots for cyclic AMP phosphodiesterase with an apparent Km for cyclic AMP of approximately 20 muM were obtained. Modification of the apparent Km of BHK cell phosphodiesterase was detectable within 20 minutes after dillution of cells grown in 10% serum into fresh medium containing 1% serum. With the BHK cell line transformed with Rous sarcoma virus, differences in enzyme kinetics were not seen when these cells were diluted in 1% or 10% serum. In addition to the serum induced differences in the apparent Km of cyclic AMP phosphodiesterases of BHK cells, total cyclic AMP and cyclic GMP phosphodiesterase activities were also modified by growth conditions. BHK cells grown to high cell densities had three to five-fold higher total cyclic AMP activity than did the cells in less dense cultures. When the dense cell cultures were diluted into fresh medium containing 10% serum, total enzyme activities fell to levels comparable to those found in the rapidly growing cells at low cell densities. The reduction in total enzyme activity after dilution of BHK cells occurred rapidly and was influenced by cell density. A similar reduction of total enzyme activity was also seen in diluted RSV cells; however, the time course of the response differed from that seen in the untransformed cells.     

Monoclonal antibody #3-9-16 recognizes one of the two isoforms of rabies virus matrix protein that exposes its N-terminus on the virion surface

We investigated behaviors of the rabies virus matrix (M) protein using a monoclonal antibody (mAb), #3-9-16, that recognized a linear epitope located at the N-terminus of the protein. Based on the reactivity with this mAb, M proteins could be divided into at least two isoforms; an ordinary major form (Malpha) whose 3-9-16 epitope is hidden, and an N-terminal-exposed epitope-positive form (Mbeta). The Mbeta protein accounted for about 25-30% of the total M proteins in the virion, while its content in the cell ranged from 10 to 15% of total M protein. Fluorescent antibody (FA) staining showed that the Mbeta antigen distributed in the Golgi area where the colocalized viral glycoprotein antigen was also detected. Mbeta antigen was shown to be exposed on the surface of infected cells by both immunoprecipitation and FA staining with the mAb, whereby the cells might have become sensitive to the mAb-dependent complement-mediated cytolysis. Similarly, the Mbeta antigen was shown to be exposed on the virion surface, and the infectivity of the virus was destroyed by the mAb in the presence of a complement. Together with these results, we think that the M protein molecule takes either of two conformations, one (Mbeta) of which exposes the 3-9-16 epitope located in the N-terminal region of the M protein, that are also exposed on the surface of the virion and infected cells, whereby it might play a certain important role(s) in the virus replication process differently from the other form (Malpha), probably through its intimate association with the Golgi area and/or the cell membrane.     

Biogenesis of type I cytopathic vacuoles in Semliki Forest virus-infected BHK cells.

We investigated the biogenesis of type I cytopathic vacuoles (CPVIs) in Semliki Forest virus (SFV)-infected cells by immunofluorescence and electron microscopy. By using the ts1 mutant of SFV at the restrictive temperature to avoid superinfection, we showed that the multiplicity of infection affects the time of appearance but not the number of CPVIs in a cell. Formation of CPVIs did not require incoming virus particles, because they were found in BHK cells transfected with infectious RNA from the SFV prototype strain or ts1 mutant. When the SFV gene for nsP3 was expressed alone in BHK cells, the nsP3 protein was localized to numerous vesiclelike structures and large vacuoles. The nsP3 protein may function as an anchoring protein for the RNA replication complex of SFV.     

Movement of interphase Golgi apparatus in fused mammalian cells and its relationship to cytoskeletal elements and rearrangement of nuclei

Virus-induced Vero cell fusion was used to analyze the rearrangement of Golgi apparatus during the development of syncytia. Individual Golgi apparatus, associated initially with the separate microtubule-organizing centers in the perinuclear area of fused cells, congregated in the center of the syncytia and formed an extended Golgi complex within 3 to 5 h. The relocation of the Golgi apparatus, but not of nuclei, depended on the presence of an intact microtubule network, since both the microtubule depolymerizing drug nocodazole and the microtubule-stabilizing drug taxol interfered with the formation of an extended Golgi complex. Depolymerization of microfilaments with cytochalasin D and the complete collapse of intermediate filaments induced by microinjected monoclonal antibodies against vimentin had no effect on these processes. Cooling cells to 20 degrees C inhibited both congregation of Golgi apparatus and relocation of nuclei. Visualization of the movement of Golgi apparatus labeled in living cells with fluorescent metabolites of C6-NBD-ceramide showed that relocation of the Golgi apparatus was a process in which congregation and coalescence of the intact organelles was seen, rather than dispersal and reassembly of smaller Golgi elements in the center of the polykaryons. Thus, movement of intact Golgi apparatus in fused interphase cells depends on an undisturbed microtubule network and occurs independently of the relocation of nuclei.     

Role of the p53 protein in cell proliferation as studied by microinjection of monoclonal antibodies.

Two monoclonal antibodies against the p53 protein, PAb 122 and 200-47, were microinjected into mammalian cells as a probe to determine the role of the p53 protein in cell proliferation. PAb 122 recognizes the p53 proteins of mouse and human cells but not of hamster cells, whereas 200-47 recognizes the p53 proteins of mouse and hamster cells but not of human cells. The ability of these antibodies to inhibit serum-stimulated DNA synthesis of cells in culture correlates with their ability to recognize the species-specific antigenic determinants. More important, however, is the observation that microinjected PAb 122 inhibits the transition of Swiss 3T3 cells from G0 to S phase, but has no effect on the progression of these cells from mitosis to the S phase.     

Microinjection of mitotic cells with the 3F3/2 anti-phosphoepitope antibody delays the onset of anaphase

The transition from metaphase to anaphase is regulated by a checkpoint system that prevents chromosome segregation in anaphase until all the chromosomes have aligned at the metaphase plate. We provide evidence indicating that a kinetochore phosphoepitope plays a role in this checkpoint pathway. The 3F3/2 monoclonal antibody recognizes a kinetochore phosphoepitope in mammalian cells that is expressed on chromosomes before their congression to the metaphase plate. Once chromosomes are aligned, expression is lost and cells enter anaphase shortly thereafter. When microinjected into prophase cells, the 3F3/2 antibody caused a concentration-dependent delay in the onset of anaphase. Injected antibody inhibited the normal dephosphorylation of the 3F3/2 phosphoepitope at kinetochores. Microinjection of the antibody eliminated the asymmetric expression of the phosphoepitope normally seen on sister kinetochores of chromosomes during their movement to the metaphase plate. Chromosome movement to the metaphase plate appeared unaffected in cells injected with the antibody suggesting that asymmetric expression of the phosphoepitope on sister kinetochores is not required for chromosome congression to the metaphase plate. In antibody-injected cells, the epitope remained expressed at kinetochores throughout the prolonged metaphase, but had disappeared by the onset of anaphase. When normal cells in metaphase, lacking the epitope at kinetochores, were treated with agents that perturb microtubules, the 3F3/2 phosphoepitope quickly reappeared at kinetochores. Immunoelectron microscopy revealed that the 3F3/2 epitope is concentrated in the middle electronlucent layer of the trilaminar kinetochore structure. We propose that the 3F3/2 kinetochore phosphoepitope is involved in detecting stable kinetochore-microtubule attachment or is a signaling component of the checkpoint pathway regulating the metaphase to anaphase transition.     

Monoclonal antibodies as markers of the endocytic and secretory pathways

A galactosyltransferase-rich subcellular fraction and wheat germ agglutinin(WGA)-binding microsomal proteins from rat myeloma cells have been used to immunize BALB/c mice. Fusion of the corresponding spleen cells with the Sp2/0 mouse myeloma has lead to the production of hybridomas secreting monoclonal antibodies directed against four proteins of the Golgi complex (GC) and other smooth membranes (SM). Subcellular fractionation of myeloma cells and rat liver, Triton X-114 partitioning, protease treatment and lectin binding studies have permitted us to identify--by immunoblotting--the molecular weight of the proteins involved, their topology and their mode of association with membranes. Morphological analysis has been performed by immunocytochemistry at the light and electron microscopic level. Judging by these criteria, the GCII antigen is a protein of 44 kDa which is loosely associated with the endodomain of Golgi cisternae. GCIII is a detergent-binding glycoprotein of 130 kDa whose epitope is on the endodomain of Golgi cisternae. SMI is a detergent-binding glycoprotein of 58 to 90 kDa found at several stations along the endocytic path: in coated pits, coated vesicles, endocytic vesicles, but not in lysosomes. The epitope recognized by the corresponding antibody faces the ectodomain. When this antibody is added to living cells in culture, it is rapidly internalized. SMII is a detergent-binding glycoprotein of 140 kDa. The epitope recognized is restricted to membranes of Golgi complex cisternae and multivesicular bodies. These reagents should be useful for dissection and perturbation of vesicular traffic.     

Mouse prenylated Rab acceptor is a novel Golgi membrane protein

We have cloned a mouse prenylated Rab acceptor (mPRA), which interacts with various Rab proteins in the yeast two-hybrid system. This study investigated its intracellular localization and characterized the localization signal. The mPRA was found to be an integral membrane protein that was localized to the Golgi complex at steady state as determined by confocal fluorescence microscopy. With green fluorescent protein attached to the N-terminus of mPRA, the fusion protein was expressed in BHK cells and was shown to exhibit the same Golgi localization as the native mPRA. Systematic truncations from the N- and C-termini of mPRA revealed that the entire N-terminal half (91 residues) of the protein was dispensable for the Golgi localization. In contrast, deletion of only 5 residues from the C-terminus diminished the Golgi localization of mPRA, leading to its accumulation in the ER. The data indicate that the C-terminal half (94 residues) of mPRA is necessary and sufficient for proper folding, ER export, and Golgi localization. The Golgi localization of mPRA suggests that it may play a role in the structural organization and function of the Golgi complex.     

Release of the glucose‐regulated protein 94 by baby hamster kidney cells

Glucose‐regulated protein 94 (grp94) is a major component of the endoplasmic reticulum (ER) lumen of eukaryotic cells. We showed that grp94 is released from baby hamster kidney (BHK‐21) cells into a serum‐free medium. The exit of grp94 into the medium was not related to the protein discharge due to cell death and was independent of de novo protein synthesis. The treatment of cells with brefeldin A and monensin, the inhibitors of the classical pathway of protein secretion, did not decrease the extracellular level of grp94, indicating that the discharge of grp94 from cells does not occur through the ER/Golgi–dependent pathway. Exosomes, membrane vesicles secreted by several cell types, were not involved in the release of grp94 from cells. Methyl‐β‐cyclodextrin, a substance that disrupts the lipid raft organization, considerably reduced the extracellular level of grp94, indicating that lipid rafts are involved in the liberation of grp94 from BHK‐21 cells. The results suggest that BHK‐21 cells release grp94 into the serum‐free medium via the nonclassical secretory pathway in which lipid rafts play an important role. Copyright © 2012 John Wiley & Sons, Ltd.