Biomedical subjects

R Pepperkok

Publications and source records attributed to R Pepperkok.

At least 19 recordsLinked to original sources

PubMed1998

An ordered inheritance strategy for the Golgi apparatus: visualization of mitotic disassembly reveals a role for the mitotic spindle.

During mitosis, the ribbon of the Golgi apparatus is transformed into dispersed tubulo-vesicular membranes, proposed to facilitate stochastic inheritance of this low copy number organelle at cytokinesis. Here, we have analyzed the mitotic disassembly of the Golgi apparatus in living cells and provide evidence that inheritance is accomplished through an ordered partitioning mechanism. Using a Sar1p dominant inhibitor of cargo exit from the endoplasmic reticulum (ER), we found that the disassembly of the Golgi observed during mitosis or microtubule disruption did not appear to involve retrograde transport of Golgi residents to the ER and subsequent reorganization of Golgi membrane fragments at ER exit sites, as has been suggested. Instead, direct visualization of a green fluorescent protein (GFP)-tagged Golgi resident through mitosis showed that the Golgi ribbon slowly reorganized into 1-3-micron fragments during G2/early prophase. A second stage of fragmentation occurred coincident with nuclear envelope breakdown and was accompanied by the bulk of mitotic Golgi redistribution. By metaphase, mitotic Golgi dynamics appeared to cease. Surprisingly, the disassembly of mitotic Golgi fragments was not a random event, but involved the reorganization of mitotic Golgi by microtubules, suggesting that analogous to chromosomes, the Golgi apparatus uses the mitotic spindle to ensure more accurate partitioning during cytokinesis.

Animals↗

PubMed1998

Three distinct steps in transport of vesicular stomatitis virus glycoprotein from the ER to the cell surface in vivo with differential sensitivities to GTP gamma S.

Microinjected GTP gamma S revealed three distinct steps in the exocytic transport of the temperature sensitive glycoprotein of vesicular stomatitis virus (ts-O45-G) from the ER to the cell surface in intact Vero cells. While COPII dependent export of ts-O45-G from the ER is blocked in cells injected with recombinant protein of a dominant mutant of SAR1a (SAR1a[H79G]) inhibited in GTP hydrolysis, neither injected GTP gamma S nor antibodies against beta-COP (anti-EAGE) interfere with this transport step significantly. In contrast, transport to the Golgi complex is blocked by 50 microM GTP gamma S, a dominant mutant of ARF1 (ARF1[Q71L]) inhibited in GTP hydrolysis, or microinjected anti-EAGE, but injected Sar1a[H79G]p has no effect. Microinjection of GTP gamma S or expression of ARF[Q71L] rapidly induces accumulation of COPI coated vesicular structures lacking ts-O45-G. Finally, transport of ts-O45-G from the trans-Golgi network (TGN) to the cell surface is inhibited only by high concentrations of GTP gamma S (500 microM). Interestingly, this step is only partially brefeldin A sensitive, and injected antibodies against beta-COP and p200/myosin II, a TGN membrane associated protein, have no effect. These data provide first strong in vivo evidence for at least three distinct steps in the exocytic pathway of mammalian cells regulated by different sets of GTPases and coat proteins. COPII, but not COPI, is required for ER export of ts-O45-G. COPI plays a role in subsequent transport to the Golgi complex, and a so far unidentified GTP gamma S sensitive coat appears to be involved in transport from the TGN to the cell surface.

Animals↗

PubMed1997

Involvement of the transmembrane protein p23 in biosynthetic protein transport.

Here, we report the localization and characterization of BHKp23, a member of the p24 family of transmembrane proteins, in mammalian cells. We find that p23 is a major component of tubulovesicular membranes at the cis side of the Golgi complex (estimated density: 12,500 copies/micron2 membrane surface area, or approximately 30% of the total protein). Our data indicate that BHKp23-containing membranes are part of the cis-Golgi network/intermediate compartment. Using the G protein of vesicular stomatitis virus as a transmembrane cargo molecule, we find that p23 membranes are an obligatory station in forward biosynthetic membrane transport, but that p23 itself is absent from transport vesicles that carry the G protein to and beyond the Golgi complex. Our data show that p23 is not present to any significant extent in coat protein (COP) I-coated vesicles generated in vitro and does not colocalize with COP I buds and vesicles. Moreover, we find that p23 cytoplasmic domain is not involved in COP I membrane recruitment. Our data demonstrate that microinjected antibodies against the cytoplasmic tail of p23 inhibit G protein transport from the cis-Golgi network/ intermediate compartment to the cell surface, suggesting that p23 function is required for the transport of transmembrane cargo molecules. These observations together with the fact that p23 is a highly abundant component in the intermediate compartment, lead us to propose that p23 contributes to membrane structure, and that this contribution is necessary for efficient segregation and transport.

Amino Acid Sequence↗

PubMed1997

Visualization of ER-to-Golgi transport in living cells reveals a sequential mode of action for COPII and COPI.

Exocytic transport from the endoplasmic reticulum (ER) to the Golgi complex has been visualized in living cells using a chimera of the temperature-sensitive glycoprotein of vesicular stomatitis virus and green fluorescent protein (ts-G-GFP[ct]). Upon shifting to permissive temperature, ts-G-GFP(ct) concentrates into COPII-positive structures close to the ER, which then build up to form an intermediate compartment or transport complex, containing ERGIC-53 and the KDEL receptor, where COPII is replaced by COPI. These structures appear heterogenous and move in a microtubule-dependent manner toward the Golgi complex. Our results suggest a sequential mode of COPII and COPI action and indicate that the transport complexes are ER-to-Golgi transport intermediates from which COPI may be involved in recycling material to the ER.

Animals↗

PubMed1997

Partitioning of the Golgi apparatus during mitosis in living HeLa cells.

The Golgi apparatus of HeLa cells was fluorescently tagged with a green fluorescent protein (GFP), localized by attachment to the NH2-terminal retention signal of N-acetylglucosaminyltransferase I (NAGT I). The location was confirmed by immunogold and immunofluorescence microscopy using a variety of Golgi markers. The behavior of the fluorescent Golgi marker was observed in fixed and living mitotic cells using confocal microscopy. By metaphase, cells contained a constant number of Golgi fragments dispersed throughout the cytoplasm. Conventional and cryoimmunoelectron microscopy showed that the NAGT I-GFP chimera (NAGFP)-positive fragments were tubulo-vesicular mitotic Golgi clusters. Mitotic conversion of Golgi stacks into mitotic clusters had surprisingly little effect on the polarity of Golgi membrane markers at the level of fluorescence microscopy. In living cells, there was little self-directed movement of the clusters in the period from metaphase to early telophase. In late telophase, the Golgi ribbon began to be reformed by a dynamic process of congregation and tubulation of the newly inherited Golgi fragments. The accuracy of partitioning the NAGFP-tagged Golgi was found to exceed that expected for a stochastic partitioning process. The results provide direct evidence for mitotic clusters as the unit of partitioning and suggest that precise regulation of the number, position, and compartmentation of mitotic membranes is a critical feature for the ordered inheritance of the Golgi apparatus.

Cells, Cultured↗

PubMed1997

Dissociation of coatomer from membranes is required for brefeldin A-induced transfer of Golgi enzymes to the endoplasmic reticulum.

Addition of brefeldin A (BFA) to mammalian cells rapidly results in the removal of coatomer from membranes and subsequent delivery of Golgi enzymes to the endoplasmic reticulum (ER). Microinjected anti-EAGE (intact IgG or Fab-fragments), antibodies against the "EAGE"-peptide of beta-COP, inhibit BFA-induced redistribution of beta-COP in vivo and block transfer of resident proteins of the Golgi complex to the ER; tubulo-vesicular clusters accumulate and Golgi membrane proteins concentrate in cytoplasmic patches containing beta-COP. These patches are devoid of marker proteins of the ER, the intermediate compartment (IC), and do not contain KDEL receptor. Interestingly, relocation of KDEL receptor to the IC, where it colocalizes with ERGIC53 and ts-O45-G, is not inhibited under these conditions. While no stacked Golgi cisternae remain in these injected cells, reassembly of stacks of Golgi cisternae following BFA wash-out is inhibited to only approximately 50%. Mono- or divalent anti-EAGE stabilize binding of coatomer to membranes in vitro, at least as efficiently as GTP(gamma)S. Taken together these results suggest that enhanced binding of coatomer to membranes completely inhibits the BFA-induced retrograde transport of Golgi resident proteins to the ER, probably by inhibiting fusion of Golgi with ER membranes, but does not interfere with the disassembly of the stacked Golgi cisternae and recycling of KDEL receptor to the IC. These results confirm our previous results suggesting that COPI is involved in anterograde membrane transport from the ER/IC to the Golgi complex (Pepperkok et al., 1993), and corroborate that COPI regulates retrograde membrane transport between the Golgi complex and ER in mammalian cells.

Animals↗

PubMed1996

Modification of the cytoplasmic domain affects the subcellular localization of Golgi glycosyl-transferases.

Our goal was to engineer a Golgi glycosyltransferase epitope-tagged on its cytoplasmically exposed, short, N-terminal domain that gave normal subcellular localization. Partial replacement of the cytoplasmic tail of human alpha-2,6-sialyltransferase (SialylT) with the negatively charged myc or FLAG epitope resulted in almost complete mislocalization of the chimera expressed in Vero cells. A granular cytoplasmic staining pattern was seen by immunofluorescence. Spacing the negatively charged residues progressively outward from the negative N-terminus resulted in increasingly more normal localization of myc or FLAG-tagged protein to a juxtanuclear Golgi-like distribution. Substitution of a neutrally charged VSV-G sequence for these tags resulted in normal localization of the chimera to the juxtanuclear Golgi region. Insertion of the myc epitope within the N-terminal domain of the short form of bovine beta-1,4-galactosyltransferase (GalT) gave a chimeric protein that mislocalized in BHK cells. No signal was detected with a monoclonal anti-epitope antibody indicating that the myc epitope was masked. Placement of myc or FLAG epitopes at the NH2-terminus of human N-acetylglucosaminyltransferase I (GlcNAc-T) resulted in chimeric proteins that in Vero cells displayed little Golgi localization. We conclude that positioning of negative charge, in particular, close to the membrane, typically produces a failure of type II Golgi glycosyltransferases to exit the ER/CGN, presumably due to quality control mechanisms. These proteins may be successfully epitope-tagged on their N-terminal domain either using a neutral or positively charged sequence or spacing any negatively charged sequence out from the membrane.

Amino Acid Sequence↗

PubMed1995

COPs regulating membrane traffic.

Cytosolic coat proteins (COPs) regulate membrane traffic in eukaryotic cells. Three classes of coat protein complexes have so far been identified: clathrin and its adaptor proteins, coatomer (COPI), and COPII. Coatomer (composed of seven different subunits) and ADP-ribosylation factor (ARF), which form the COPI coat, are required for budding of coated vesicles from membranes. COPI has been implicated in several steps of transport from the intermediate compartment to the cis-Golgi network, through cisternae of the Golgi stack, and is essential for retrieval to the endoplasmic reticulum (ER) of membrane proteins containing the carboxy-terminal dilysine ER-retention motif. A family of structurally and functionally related COPs may regulate all membrane traffic steps in eukaryotic cells.

ADP-Ribosylation Factors↗

PubMed1995

Immunocytochemical localization of beta-COP to the ER-Golgi boundary and the TGN.

Recent data strongly suggest that the coatomer (COP) complex is involved in membrane transport between the ER and Golgi complex. This vesicular coat has been implicated in ER to Golgi, in intra Golgi as well as in Golgi to ER traffic. In this study we present a detailed immunocytochemical analysis of the distribution of beta-COP in different tissue culture cells. Our results extend previous studies by showing, using electron microscopy, that beta-COP accumulates on vesicular profiles and buds in the intermediate compartment (IC) under conditions that block ER to Golgi transport (15 degrees C). Importantly, under these conditions beta-COP co-localizes on these structures with a passenger protein, the membrane glycoprotein of vesicular stomatis virus (ts-O45-G). Furthermore, quantitative immunofluorescence microscopy of cells with ts-045-G accumulated in the ER, IC and trans-Golgi network, shifted briefly to the permissive temperature, showed that beta-COP was associated with many of the putative transport intermediates containing the viral glycoprotein which is in transit between the ER/IC and the cis-Golgi. The simplest interpretation of these data is that COP-coated vesicles are involved in anterograde transport of ts-045-G from the IC to the Golgi complex. Since many putative COP vesicle lacked the G protein following release of the 15 degrees C block this pool could be involved in retrograde transport. We also show that beta-COP is present on the membranes of the trans-Golgi network. However, in contrast to the ER-Golgi boundary, we could find no convincing evidence that this pool of beta-COP is associated with buds or trans-Golgi network-derived transport vesicles.

Animals↗

PubMed1994

Regulation of G1 progression by E2A and Id helix-loop-helix proteins.

In NIH3T3 fibroblasts, the ubiquitous helix-loop-helix (HLH) protein E2A (E12/E47) and the myogenic HLH proteins MyoD, MRF4 and myogenin are growth-inhibitory, while two ubiquitous Id proteins lacking the basic region are not. The dimerization domain mediates inhibition. However, in addition to the HLH region, E2A contains two inhibitory regions over-lapping with the main transcriptional activation domains. The growth-suppressive activity of the intact E47 as well as MyoD was counteracted by the Id proteins. When E47 lacking the HLH domain was overexpressed, Id could no longer reverse growth inhibition. By increasing the amount of E47 with an inducible system or neutralizing the endogenous Id with microinjected anti-Id antibodies, withdrawal from the cell cycle occurred within hours before the G1-S transition point. The combined results suggest that the Id proteins are required for G1 progression. The antagonism between the E2A and Id proteins further suggests that both are involved in regulatory events prior to or near the restriction point in the G1 phase of the cell cycle.

3T3 Cells↗

PubMed1994

Id proteins control growth induction in mammalian cells.

Id1, Id2, and Id3 (HLH462) dimerize with members of the basic helix-loop-helix protein family, but due to the absence of the basic region, the resulting heterodimers cannot bind DNA. Therefore Id-type proteins negatively regulate DNA binding of the basic helix-loop-helix proteins. Here we report that Id1, Id2, and Id3 are induced shortly after serum stimulation in arrested NIH 3T3. Antisense oligonucleotides against the Id mRNAs delay the reentry of arrested cells into the cell cycle elicited by stimulation with serum or growth factors. Antisense oligonucleotides against all three Id mRNAs are more effective than individual ones. Combined, these results indicate that Id proteins are involved in the control of growth induction.

3T3 Cells↗

PubMed1994

Casein kinase II is required for transition of G0/G1, early G1, and G1/S phases of the cell cycle.

Casein kinase II (CKII) is a ubiquitous serine/threonine protein kinase with many cellular functions, including participation in mitogenic signaling by cytoplasmic nuclear translocation (Lorenz, P., Pepperkok, R., Ansorge, W., and Pyerin, W. (1993) J. Biol. Chem. 268, 2733-2739). To examine whether cell compartment-specific availability is a requirement for CKII function during cell cycle progression, antibodies against CKII beta, the regulatory subunit of CKII, were microinjected into the cytoplasm or the nucleus of G0-synchronized human primary fibroblasts (IMR-90) at the time of mitogenic stimulation or at various intervals thereafter. Significant inhibition of the stimulation was obtained with both cytoplasmic and nuclear injections. The inhibition was reversible, was not observed with control antibodies, and was abolished by co-injection of purified CKII holoenzyme. The inhibition differed, however, in extent, duration, and cell cycle phase between cytoplasmic and nuclear injections. After cytoplasmic injection, inhibition reached 45-50% and was effective at two intervals within the first 2 h and at 12-16 h post-stimulation, i.e. at G0/G1 phase transition and at the G1/S phase boundary of the cell cycle. After injection into the nucleus, the inhibition was considerably stronger, reaching 80-85%, and was effective for the first 6 h post-stimulation, i.e. for the transition of G0/G1 phase and the adjoining first part of G1 phase. Cytoplasmic or nuclear injections within S phase affected neither DNA synthesis nor cell division. The data suggest that cell cycle transition from G0 to S phase requires the presence of a certain functional level of CKII at defined times and at defined cellular locations as follows: for transition of G0/G1 at both the nucleus and the cytoplasm, for transition of early G1 at the nucleus, and for transition of G1/S at the cytoplasm.

Animals↗

PubMed1994

Coat proteins in intracellular membrane transport.

Transport of newly synthesized material from the endoplasmic reticulum (ER) towards the Golgi complex, through the Golgi cisternae, and out of the trans-Golgi network (TGN) is thought to be mediated by vesicular carriers. Different types of vesicle are involved in this biosynthetic membrane traffic. All are coated with protein complexes on their cytoplasmic surface. COP-coated vesicles have recently been implicated in transport of cargo from the ER to the TGN, and clathrin-coated vesicles from the TGN to endosomes, but the carriers moving material to the cell surface are still unknown. Sequence homologies between subunits of the COP- and the clathrin-adaptor complexes suggest that coat proteins may belong to a family of proteins with related functions. The precise role of the coat proteins is not fully understood, although they have been implicated in clustering of cargo into buds and in budding of vesicles. In addition, coat proteins may play an essential role in targeting of transport intermediates and may serve to regulate membrane fusion.

Animals↗

PubMed1994

A human nuclear protein with sequence homology to a family of early S phase proteins is required for entry into S phase and for cell division.

Molecular cloning and characterisation of a human nuclear protein designated BM28 is reported. On the amino acid level this 892 amino acid protein, migrating on SDS-gels as a 125 kDa polypeptide, shares areas of significant similarity with a recently defined family of early S phase proteins. The members of this family, the Saccharomyces cerevisiae Mcm2p, Mcm3p, Cdc46p/Mcm5p, the Schizosaccharomyces pombe Cdc21p and the mouse protein P1 are considered to be involved in the onset of DNA replication. The highest similarity was found with Mcm2p (42% identity over the whole length and higher than 75% over a conservative region of 215 amino acid residues), suggesting that BM28 could represent the human homologue of the S. cerevisiae MCM2. Using antibodies raised against the recombinant BM28 the corresponding antigen was found to be localised in the nuclei of various mammalian cells. Microinjection of anti-BM28 antibody into synchronised mouse NIH3T3 or human HeLa cells presents evidence for the involvement of the protein in cell cycle progression. When injected in G1 phase the anti-BM28 antibody inhibits the onset of subsequent DNA synthesis as tested by the incorporation of bromodeoxyuridine. Microinjection during the S phase had no effect on DNA synthesis, but inhibits cell division. The data suggest that the nuclear protein BM28 is required for two events of the cell cycle, for the onset of DNA replication and for cell division.

3T3 Cells↗

PubMed1994

The intracellular mobility of a viral membrane glycoprotein measured by confocal microscope fluorescence recovery after photobleaching.

Fluorescence recovery after photobleaching (FRAP) has been a powerful tool for characterizing the mobility of cell surface membrane proteins. However, the application of FRAP to the study of intracellular membrane proteins has been hampered by the lack of specific probes and their physical inaccessibility in the cytoplasm. We have measured the mobility of a model transmembrane protein, the temperature-sensitive vesicular stomatitis viral membrane glycoprotein (ts-O45-G), in transit from the endoplasmic reticulum (ER) to the Golgi complex. ts-O45-G accumulates in the ER at nonpermissive temperature (39.5 degrees C) and is transported via the Golgi complex to the surface upon shifting cells to the permissive temperature (31 degrees C). Rhodamine-labeled Fab fragments against a cytoplasmic epitope of ts-O45-G (rh-P5D4-Fabs) were microinjected into cells to visualize the intracellular viral membrane protein and to determine its mobility by FRAP with a confocal microscope. Moreover, we have measured the effects of microinjected antibodies against beta-COP on the mobility of ts-O45-G following release of the temperature block. FRAP was essentially complete when rh-P5D4-Fab-injected cells were bleached either following release of labeled ts-O45-G from the ER or upon its accumulation at 20 degrees C in the trans-Golgi network (TGN). In contrast, recovery was reduced by about one third when infected cells had been injected with antibodies that bind to beta-COP in vivo. The diffusion constant of mobile ts-O45-G under all conditions was approximately 10 x 10(-10) cm2/s. These results validate the feasibility of FRAP for the study of an intracellular transmembrane protein and provide the first evidence that such a protein is highly mobile.

Animals↗

PubMed1994

Molecular characterization of two functional domains of CLIP-170 in vivo.

CLIP-170 is a microtubule-binding protein isolated from HeLa cells that is involved in the interaction of endosomes with microtubules. The basic N-terminal domain of CLIP-170 binds to microtubules in vitro. To characterize further the functional domains of this cytoplasmic linker protein, we have transiently expressed intact and mutant forms of CLIP-170 in mammalian cells (HeLa and Vero cells) and show that the tandem repeat present in the N-terminal domain is essential for its binding to microtubules in vivo as previously found in vitro. With increasing levels of expression of CLIP-170, the sites with which the peripheral ends of microtubules interact enlarge, eventually forming large patches, which finally lead to the apparent bundling of microtubules. These patches do not form when the C-terminal domain is absent from the transfected protein. Modification of the microtubule-binding region, particularly of the tandem repeat motif, modulates the binding of CLIP-170 to microtubules. Overexpressed CLIP-170 appears neither to interact with nor to influence the organization of the intermediate filaments, and collapsing the network of intermediate filaments with microinjected antibodies against vimentin has no effect on the distribution of CLIP-170. These data suggest that CLIP-170 has at least two functional domains in vivo, an N-terminal microtubule-binding domain, and a C-terminal domain that is involved in the anchoring of microtubules to peripheral cytoplasmic structures.

Amino Acid Sequence↗

PubMed1994

Requirement of casein kinase 2 for entry into and progression through early phases of the cell cycle.

Requirement of protein kinase CK2 during cell cycle was examined by specific perturbation of CK2 in the intact cell by antisense-oligodeoxynucleotides and microinjection of antibodies. When quiescent human primary lung fibroblasts (IMR-90) were exposed before growth stimulation to oligodeoxynucleotides complementary to the translation start region of mRNAs encoding subunit alpha or beta, a significant inhibition of growth stimulation by epidermal growth factor or serum was observed. The inhibition was reversible and decreased or abolished with mutated antisense-oligodeoxynucleotides. The inhibitory effect coincided with a decrease of CK2 protein (immunostaining with beta subunit antibody) at entry into and during the first several hours of the cell cycle. Injection of beta-specific monoclonal and polyclonal antibodies into IMR-90 cells caused significant inhibition of growth stimulation. The inhibition was reversible, not observed with control antibodies, and strongly reduced by coinjection of CK2 holoenzyme. Cytoplasmic injection inhibited up to 50-60% and was effective at two intervals within the first 2 h and at 12-16 h poststimulation, i.e., at G0/G1 phase transition and at G1/S boundary, respectively. The inhibition at G0/G1 transition is paralleled by an inhibition of cytoplasmic-nuclear translocation of beta subunit protein. Injection of beta antibodies into the nucleus inhibited growth stimulation by as much as 80-85% and was effective for the first 6 h poststimulation, i.e., at G0/G1 phase transition and progression through the adjoining early G1 phase. Nuclear as well as cytoplasmic injections performed during S phase affected neither DNA synthesis nor cell division.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗

PubMed1993

The Src family tyrosine kinases are required for platelet-derived growth factor-mediated signal transduction in NIH 3T3 cells.

Three members of the Src family of protein tyrosine kinases Src, Fyn, and Yes associate with the activated platelet-derived growth factor (PDGF) receptor in vivo. This interaction requires the Src homology 2 (SH2) domain of the Src family member and causes activation of the intrinsic activity of the Src family kinases. We microinjected cells with DNA encoding catalytically inactive forms of the Src and Fyn proteins and examined their effects on PDGF-mediated signaling in vivo. Kinase-inactive Src and Fyn inhibited PDGF-stimulated entry of cells into S phase, whereas kinase-active forms of the proteins had no inhibitory effects. An intact SH2 domain was required for inhibition. Furthermore, when kinase-inactive Fyn was comicroinjected with a plasmid expressing activated Ras, the cells could enter S phase, indicating that the expression of kinase-inactive Fyn did not damage cell viability. Injection of an antibody specific for Src, Fyn, and Yes also reduced signal transduction through the PDGF receptor but only when injected within 8 hr of PDGF stimulation. Together these results indicate that the ubiquitously expressed Src family members are required for PDGF-induced mitogenic signaling.

3T3 Cells↗