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Multipronged interaction of the COG complex with intracellular membranes a
a
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Rose Willett , Irina Pokrovskaya , Tetyana Kudlyk & Vladimir Lupashin a
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Department of Physiology and Biophysics; UAMS; Little Rock, AR USA
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Tomsk State University; Tomsk, Russian Federation Published online: 13 Feb 2014.
Click for updates To cite this article: Rose Willett, Irina Pokrovskaya, Tetyana Kudlyk & Vladimir Lupashin (2014) Multipronged interaction of the COG complex with intracellular membranes, Cellular Logistics, 4:1, e27888, DOI: 10.4161/cl.27888 To link to this article: http://dx.doi.org/10.4161/cl.27888
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Research Paper
Research Paper
Cellular Logistics 4, e27888; February; © 2014 Landes Bioscience
Multipronged interaction of the COG complex with intracellular membranes Rose Willett1, Irina Pokrovskaya1, Tetyana Kudlyk1 and Vladimir Lupashin1,2,* Department of Physiology and Biophysics; UAMS; Little Rock, AR USA; 2Tomsk State University; Tomsk, Russian Federation
1
Keywords: COG complex, Golgi, vesicular trafficking, vesicular tethers, intra-Golgi transport, SNARE, p115, COPI, membrane binding
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Abbreviations: COG, conserved oligomeric Golgi; mCh, mCherry; SNARE, soluble N-ethylmalemide-sensitive fusion attachment protein receptor
The conserved oligomeric Golgi complex is a peripheral membrane protein complex that orchestrates the tethering and fusion of intra-Golgi transport carriers with Golgi membranes. In this study we have investigated the membrane attachment of the COG complex and it’s on/off dynamic on Golgi membranes. Several complimentary approaches including knock-sideways depletion, FRAP, and FLIP revealed that assembled COG complex is not diffusing from Golgi periphery in live HeLa cells. Moreover, COG subunits remained membrane-associated even in COG4 and COG7 depleted cells when Golgi architecture was severely affected. Overexpression of myc-tagged COG sub-complexes revealed that different membrane-associated COG partners including β-COP, p115 and SNARE STX5 preferentially bind to different COG assemblies, indicating that COG subunits interact with Golgi membranes in a multipronged fashion.
Introduction The transport of vesicular carriers in the secretory pathway is facilitated by specialized proteins and protein complexes that assist in formation, movement, tethering, and fusion of trafficking intermediates.1 While several core components of vesicle trafficking machinery including cargo receptors, SNAREs and small GTPases are stably associated with cellular membranes via trans-membrane domains or lipid moieties, other components, including coat proteins, molecular motors and tethering factors, transiently interact with trafficking intermediates and target membranes. In order to achieve both high rate and high specificity of vesicle delivery, membrane attachment of these factors has to be tightly regulated in both a spatial and temporal manner. The conserved oligomeric Golgi (COG) complex belongs to a diverse family of vesicular tethering factors that assist in tethering and fusion of intracellular trafficking intermediates with acceptor membranes in both secretory and endocytic pathways.2-5 The COG complex is comprised of 8 subunits, named COG1–86,7 which are divided into two subcomplexes, lobe A (COG1–4) and lobe B (COG5–8).8,9 Vesicle tethering factors are thought to interact with both vesicular and target membranes through the association with small GTPases, SNAREs, coat proteins, and membrane phospholipids, but in many cases their exact mode of membrane attachment and membrane on-off dynamics remained unknown. Several Golgi tethering factors including giantin and golgin-84 are permanently associated with membranes
via transmembrane domains10,11 while other coiled-coil tethers including p115 rapidly cycle on-off Golgi membrane.12 The membrane association of the subclass of membrane tethers termed the multisubunit tethering complexes (MTC’s),5 of which the COG complex is a member of, is more complicated. The MTC’s are all peripherally associated with membranes through interactions with vesicle targeting/fusion machinery. Intriguingly, the Exocyst complex has been shown to directly interact with Rho GTPases, SNAREs, as well as PI(4,5)P2 through two of its subunits Exo7 and Sec3 to facilitate its membrane attachment.13-15 Likewise with the COG complex, we have demonstrated how interaction of the COG6 subunit with the Golgi SNARE STX6 is critical for COG6 localization on membranes.16 The COG complex is also a known Rab effector, interacting with several different key Golgi Rab proteins including the yeast Ypt1 and Ypt6,17 and the mammalian Rab1a, Rab4a, Rab6a, and Rab30.18 Finally, the COG complex will also interact with βCOP and coiled-coil tethers p115, GM130, Giantin, golgin84, TMF, and CASP.18-21 This extensive range of COG interactions with trafficking regulators provides for a multipronged method of attachment of COG subunits to Golgi and vesicle membranes. In this study we have investigated the membrane attachment of the COG complex and it’s on/off dynamic on Golgi membranes. We demonstrate that while individual COG subunits are capable of freely diffusing in a cell, once in their complex/ subcomplex, the subunits will not readily move from Golgi membranes. Next, we unveil the tendency for individual COG
*Correspondence to: Vladimir Lupashin; Email: [email protected] Submitted: 11/25/13; Accepted: 01/16/14 Citation: Willett R, Pokrovskaya I, Kudlyk T, Lupashin V. Multipronged interaction of the COG complex with intracellular membranes. Cellular Logistics 2014; 4:e27888; http://dx.doi.org/10.4161/cl.27888 www.landesbioscience.com Cellular Logistics e27888-1
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Figure 1. For figure legend, see page 3.
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Figure 1 (see previous page). Transiently expressed COG subunits are efficiently relocalized to mitochondria in a knock-sideways assay. A schematic diagram of the mitochondrial knock-sideways protein constructs (A). HeLa cells were transiently transfected with FKBP12-mCherry-ActA and GFP-FRB (B, C), COG4-GFP-FRB (D, E), or COG8-GFP-FRB (F, G). 24hrs after transfection cells were treated with 0.2μM rapamycin for 60 min. After rapamycin treatment cells were fixed and analyzed by confocal microscopy. Line plots for overlap between red and green channels are shown measuring the relative value of signal intensity (y-axis) over the distance measured in pixels (x-axis). Size bar, 10 μm.
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subunits to remain bound to Golgi membranes in a fractionation assay, despite the disturbance of the COG complex via siRNA COG depletion. Finally, we reveal a preference for COG complex/subcomplex interactions with COG interacting trafficking regulators using a complete COG complex expression model. These results demonstrate that the attachment of the COG complex to Golgi membranes is multipronged and facilitated through multiple interactions.
Results The COG complex is tightly associated with Golgi membranes To investigate the dynamics of individual COG subunits on the Golgi membrane we first applied a “knock-sideways” strategy of rapamycin-inducible capturing of soluble proteins on mitochondria membranes22. Plasmids encoding mitochondriatargeted FKBP12-mCherry-ActA as well as siRNA-resistant COG4 and COG8 with attached GFP-FRB tag were constructed (Fig. 1A). Soluble GFP-FRB was used as a positive control. Transient (less than 30 h) expression of GFP-FRB-tagged proteins revealed that, upon expression in HeLa cells, the majority of expressed proteins demonstrate a diffuse pattern, indicating soluble cytoplasmic localization (Fig. 1B, D and F). This phenotype likely indicates a delay in the incorporation of tagged COG subunits into functional Golgi-localized complexes due to a competition with the endogenous COG proteins. Indeed, longer expression demonstrated partial localization of tagged COG subunits on the Golgi membrane (data not shown). As expected, the FKBP12-mCherry-ActA construct was extensively localized to the mitochondria and addition of rapamycin to cells induced rapid and specific relocalization of all GFP-FRB tagged soluble protein to the mitochondrial vicinity (Fig. 1C, E and G). This data indicates that soluble COG subunits are freely diffusible in HeLa cells. We next wanted to investigate the dynamics of COG subunits in the assembled COG complex. Using a gene replacement strategy, we first knocked-down the endogenous COG4 and COG8 with the corresponding siRNA, and then transfected cells with plasmids expressing siRNA-resistant GFP-FRB-tagged proteins. As expected, this strategy allowed for the efficient incorporation of GFP-FRB –tagged proteins into Golgi-localized COG complexes (Fig. 2A and C). Surprisingly, upon rapamycin treatment, the assembled COG subunits failed to relocalize to FKBP12mCherry-ActA-decorated mitochondria, indicating that the COG complex is not diffusing freely from the Golgi vicinity (Fig. 2B and D). This was also evident after longer treatment, with 2, 4, and 24 h incubation with Rapamycin still unable to relocalize the GFP-FRB tagged COG subunits to the mitochondria (data not shown).
The assembled COG complex has a molecular weight ~700 KD7,17,23 and therefore may diffuse in the cytoplasm with much slower dynamics as compared with individual COG subunits. To test this hypothesis we wanted to compare the COG dynamic with that of COPI vesicle coat, a protein complex that has a similar size and is rapidly assembling/disassembling during the vesicular trafficking cycle24. siRNA-resistant GFP-FRB-tagged εCOP was properly localized in HeLa cells (Fig. 2D) and rapidly relocalized to the mitochondria decorated with FKBP12-mCherryActA (Fig. 2F), indicating that the COG complex’s size does not play a significant role in this relocalization assay. This reinforced the notion that the assembled COG complex is not diffusing away from the Golgi vicinity. We next wanted to explore the dynamic of the COG subunits in an in vivo setting. To achieve this, we performed FRAP (fluorescence recovery after photobleaching) (Fig. 3A) and FLIP (fluorescence loss in photobleaching) (Fig. 3B) assays in cells stably expressing YFP-COG3 or COG8-GFP-FRB. For both COG3 and COG8, the t1/2 for maximum recovery of fluorescence was significantly increased compared with the v-SNARE GFP-GS15 and the small GTPase YFP-Arf1 (Fig. 3A and C). This slow recovery is indicative of a lack of diffusible COG complex in the cell, and suggests that the majority of COG subunits constantly remaining in the Golgi vicinity. Moreover, the FRAP of YFP-hCOG3 had a slower recovery than hCOG8-GFP-FRB (t1/2 of 152.6s and 122s respectively) indicating that lobe A subunits are potentially more tightly retained on membranes than lobe B subunits. In line with this data, we saw that in the FLIP experiments that the loss of COG8-GFP-FRB was similar to that of v-SNARE GFP-GS15 and much slower than the YFP-Arf1, suggesting the tight association of the COG complex with Golgi membranes and/or the Golgi vicinity. COG subunit localization in COG compromised cells Having demonstrated how COG subunits once in their complex remain tightly associated with Golgi vicinity, we next wanted to explore their membrane association when the complex is partially disrupted. Using a saponin fractionation assay25 we separated WT and COG4 depleted HeLa cells into membrane and cytosol fractions, and then determined the membrane association of COG subunits 3, 4, 6, and 8. In WT cells the distribution of the COG subunits in membrane and cytosol fractions is 40%:60%, respectively (Fig. 4A and B). This is in good agreement with COG fractionation data obtained from mechanically disrupted HeLa cells26. Upon depletion of COG4, the saponinextractible pool of COG subunits was increased, but surprisingly a significant fraction (20–30%) of both lobe A subunit COG3 and lobe B subunits COG6 and COG8 was still associated with saponin-resistant membranes. Confocal microscopy revealed that COG3 remained associated with Golgi membranes in both WT and COG4-depleted cells while COG8 changed its localization
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Figure 2. COG subunits that assemble on the Golgi membrane are not available for the mitochondria capturing. HeLa cells were treated with COG4 siRNA (A, B), COG8 siRNA (C, D) or εCOP siRNA (E, F). Twenty-four hours after siRNA treatment cells were transfected with FKBP12-mCherry-ActA and COG4-GFP-FRB (A, B), COG8-GFP-FRB (C, D), or εCOP (E, F). 48 h after second transfection cells were treated with 0.2μM rapamycin for 60 min, fixed and analyzed by confocal microscopy. Line plots for overlap between red and green channels are shown measuring the relative value of signal intensity (y-axis) over the distance measured in pixels (x-axis). Size bar, 10 μm.
from the Golgi in WT cells, to a dispersed puncta-like pattern in COG4-depleted cells (Fig. 4C and D). These puncta are likely to
represent COG8’s association with Golgi-derived vesicles which are dramatically accumulated in cells upon COG4 depletion
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(compare Golgi area in electron micrograph in Fig. 4E and F, arrows). We, and others, have demonstrated previously that depletion of COG4 and COG7 in HeLa cells have the most dramatic effect on Golgi morphology and stability of Golgi-localized components of glycosylation machinery.27-29 Evaluation of COG subunit localization in COG7depleted cells revealed that the membrane association of lobe A subunits was slightly increased (Fig. 5A and B). The level of COG6 was significantly decreased in COG7-depleted cells, while the level of another lobe B subunit COG8 was not affected and remained mostly associated with the membrane fraction. Again, like in the case of COG4-depleted cells, COG8 signal was primarily found in dispersed puncta (Fig. 5C) and not associated with severely dilated and fragmented (Fig. 5D) Golgi membranes. This data indicates that even a partially destroyed COG complex, and/or remaining sub-complexes, maintain their membrane association. COG subcomplexes selectively interact with membrane bound partners To get insights into the molecular nature of the interaction of COG subcomplexes with their membrane partners, we have co- expressed preassembled lobe A, lobe B, or the complete COG complex with GFP-tagged partner proteins. In this set-up every COG subunit was labeled with the same 3xmyc tag allowing for precise quantification of COG-partner interactions. Our analysis revealed that individual lobe A and lobe B subcomplexes were capable of interaction with the COPI coat complex via the C-terminus of the βCOP subunit,18 whereas interaction of the completely assembled COG complex was significantly lower (Fig. 6A). In contrast, the coiled-coil tether p115 preferably interacted with lobe A, as well as the whole COG complex (Fig. 6B), while Golgi SNARE STX5 was preferentially binding to lobe B and the whole COG complex (Fig. 6C). This data indicates that different COG assemblies
Figure 3. Lobe A and lobe B COG subunits display different kinetic mobility profiles. HeLa cells stably expressing YFP-Arf1, GFP-GS15, YFP-hCOG3, or hCOG8-GFP-FRB were incubated at 37 °C and visualized with a LSM 510 laser confocal microscope. (A) Averaged FRAP experiments bleaching an ROI of the Golgi for 15s then measuring the recovery every 15s for 10min. (B) Averaged FLIP experiments bleaching an area in the cell periphery every 15s for 10min and measuring the signal loss for the Golgi ROI.. (C) FRAP t1/2 values. n denotes number of cells quantified. ROI, regions of interest.
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Figure 4. COG lobe B subunits lose Golgi localization in in COG4 KD cells, but remained membrane-associated. Control and 96h COG4 KD HeLa cells were treated with 0.01% saponin to fractionate into membrane and cytosol fractions (A). Fractions were separated on an SDS–PAGE gel, transferred to nitrocellulose membrane, and probed with antibodies against COG3, COG4, COG6, and COG8. Membrane and cytosol separation was measured by blotting against cytosolic GAPDH and membrane associated Vti1a proteins. Experiments were performed in triplicates. (B) Quantification of the COG subunits in the membrane associated fraction. HeLa T2-GFP control (C) and 96h COG4 KD (D) cells plated on coverslips were fixed and stained with COG3 and COG8 antibodies and analyzed by confocal microscopy. Electron micrograph of control (E) and 96h COG4 KD (F) HeLa cells fixed and processed for TEM. Arrows denote Golgi membranes, “M” denotes mitochondria. * denotes non-specific band recognized by COG6 antibodies. Error bars denote standard deviation. Size bar, 10 μm unless otherwise noted.
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Figure 5. COG subunits remain membrane associated in cells depleted for COG7. Control and 96h COG7 KD HeLa cells were treated with 0.01% saponin and fractionated into membrane and cytosol fractions (A). Fractions were separated on an SDS–PAGE gel, transferred to nitrocellulose membrane, and probed with antibodies against COG3, COG4, COG6, and COG8. Membrane and cytosol separation was measured by blotting against cytosolic GAPDH and membrane associated Vti1a. Experiments were performed in triplicates. (B) Quantification of the COG subunits in the membrane associated fraction. HeLa T2-GFP 96h COG7 KD (C) cells plated on coverslips were fixed and stained with COG3 and COG8 antibodies and analyzed by confocal microscopy. Electron micrograph 96h COG7 KD (D) HeLa cells fixed and processed for TEM. Arrows denote Golgi membranes, “M” denotes mitochondria. Error bars denote standard deviation. Size bar, 10 μm, unless otherwise noted.
possess different specificity for their membrane-bound partners. It is this multipronged interaction profile that is likely to be responsible for the tight association of the COG complex with the Golgi membranes.
Discussion In this study we illustrated how the COG complex is tightly associated with Golgi vicinity through multiple interactions with trafficking regulating components. Using a
knock-sideways approach we revealed that soluble expressed COG subunits are capable of being mis-localized to non secretory compartments, indicating that they are freely diffusible. This is in stark contrast to COG subunits once incorporated into the COG complex, and thus membrane localized, which are no longer diffusing throughout the cell. This lack of diffusion on and off of Golgi vicinity is also characterized by the strong tendency of COG subunits to remain membrane bound despite disturbances of the COG complex via depletion of individual subunits.
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Figure 6. COG subcomplexes show binding preference to different membrane-associated partners. HEK 293 cells transiently transfected with COGMyc multi-expression plasmids and GFP tagged partner proteins as indicated (lobe A expression: hCOG1/2/4–3Myc + mCOG3–3Myc; lobe B expression: hCOG6/8–3Myc + hCOG5/7–3Myc; lobe A + lobe B; hCOG1/2/4–3Myc + mCOG3–3Myc + hCOG6/8–3Myc + hCOG5/7–3Myc). (A) GFP-βCOP-CTD (aa 666–953) (B) GFP-P115 (C) GFP-STX5, and (E) GFP as a negative control for COG-Myc binding. 36 h post transfection, cells were collected and lysed, and GFP–SNARE-interacting proteins were precipitated with anti-GFP antibodies. Immuno-precipitates, along with 5% or 10% of total input, were separated on an SDS–PAGE gel, transferred to nitrocellulose membrane and probed with antibodies against Myc (upper panel) and GFP (lower panel). (F) Quantification of COG-Myc binding.
This tight membrane association of COG subunits to Golgi membranes despite depletions of individual subunits is not a new concept. We have shown that in yeast, individual COG subunit depletion still results in all subunits present in a total membrane fraction8. Additionally, primary fibroblasts from a
COG4 mutation CDG-II patient revealed that despite a lack of membrane association of the R729W COG4 point mutant, both lobe A and lobe B COG subunits still remain membrane bound.30 These results are further supportive of our conclusion that the membrane attachment of the COG complex to Golgi/
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vesicle membranes is achieved through multiple interactions with individual COG subunits. We demonstrate for the first time an expression model wherein all 8 COG subunits are expressed and pre-assembled to characterize COG interactions at a physiological and quantifiable setting. Gel filtration of HeLa cell membrane and cytosol fractions has eluded to a lack of monomeric COG subunits at steady-state (Willett and Lupashin, in progress), indicating that COG subunits are always in either a complex or subcomplex in the cell. In light of this, we developed this assay to now probe for COG interactions in their native conformations allowing for a more concise interpretation of the COG interactome.4 Indeed, we see that the expression of subcomplexes/complex adds an additional level of selectivity of COG interactions to highlight those which are more physiological. This exciting new assay urges for a confirmation of many of the individual protein-protein interactions that have been previously described for the COG complex.16,18-20,31 These results also shed new light on the steady-state membrane/ cytosol distribution of the COG complex. Previously it was published that the yeast COG complex was preferentially in a soluble pool32. This split was identified using spheroplasts that underwent harsh conditions (NaN3 pre-treatments). Alternatively, mechanically disrupted yeast and mammalian cells demonstrated preferentially membrane localization of COG subunits.26,33 In this work we have seen that the split between the membrane bound and soluble pool of the COG complex using a gentle saponin extraction protocol is closer to a 40:60 split (Fig. 4A and B). This 40:60 split may also be an over-statement of the soluble pool of the COG complex as seen with FRAP and FLIP assays (Fig. 3). In a live cell situation, we see that there is very little recovery of fluorescence after bleaching, indicating that there is not a readily available diffusible pool of COG complex, and rather that the majority of the protein is always present in the Golgi vicinity. We hypothesize that the appearance of the soluble pool in fractionation studies is due to the rapid loss of association of the COG complex with Golgi membranes upon disruption of cells. In order for the COG complex to be functional in tethering retrograde vesicles at the Golgi, it must first and foremost be properly localized to Golgi area. Our previous work has shown that one of the functions of the COG complex is to serve as a landmark for two SNARE complexes, the cis-Golgi localized STX5 containing SNARE complex, and the trans-Golgi localized STX16 containing SNARE complex.34 In order for the COG complex to organize these SNARE complexes, it is critical that they interact in the correct Golgi compartment. We believe that it is through the COGs multiple interaction profiles with membrane-bound partners (i.e., Rabs, SNAREs, coiled-coil tethers such as p115) that the compartment specificity of the COG complex is achieved. The interaction detected between the COPI coatomer and the COG subcomplexes also contributes to an interesting model of COG mediated vesicle fusion. As indicated, the βCOP subunit is interacting almost exclusively with the lobe A and lobe B subcomplexes (Fig. 6A). Therefore, we can envision a model wherein COPI coat (either on intra-Golgi vesicle or bound onto Golgi membranes) is destabilized upon interaction with COG subunits, thus uncoating the membrane and allowing for fusion. Support
for this hypothesis is found with another multisubunit tethering complex, the Dsl1 complex, which has been shown to directly interact with COPI subunits α-COP and δ-COP via Dsl1p35-37 and depletion of this Dsl1 complex subunit leads to an accumulation of COPI coated vesicles. Currently it is still unknown whether or not the COG complex has the same ability to uncoat vesicles and is a topic that requires further investigation. Given the low overall co-IP efficiencies we detect in our assays, we can also conclude that COG-membrane partner interactions are very transient and unstable in an in vitro setting. This multipronged interaction approach still raises many questions, including the time relationship of the interactions. It will be very important to know whether these interactions are occurring simultaneously or if there is a hand-off of the COG complex from the different interacting partners.
Methods Reagents and antibodies Protein G agarose was purchased from Roche. Antibodies used for immunofluorescence (IF) microscopy or western blotting (WB) were purchased through commercial sources, received as gifts from generous individual investigators, or generated by us via affinity purification. Antibodies were as follows: Rabbit polyclonal: Myc (Bethyl Laboratories); COG3, COG4, COG6, COG8 (this lab).27 Mouse monoclonal: GFP (Molecular Probes); GFP (Covance); GAPDH (Santa Cruz); Myc (Cell Signaling); Vti1a (BD Biosciences). Secondary IRDye 680 goat anti-rabbit, IRDye 700 goat anti-mouse and IRDye 800 donkey anti-goat for WB were from LI-COR Biosciences. Anti-rabbit HiLyte 488, HiLyte 555, and DyLight647 for IF were obtained from AnaSpec and Jackson ImmunoResearch, Inc. Cell culture HeLa cells were cultured in DMEM/F-12 medium (Thermo Scientific) supplemented with 15 mM HEPES, 2.5 mM L-glutamine and 10% FBS (Atlas Biologicals) at 37 °C and 5% CO2 in a 90% humidified incubator. GFP-GS15, YFP-Arf1 and COG8-GFP-FRB stable cell lines were generated by transfecting cells with corresponding plasmids (Table S1) followed by selection for G418 resistance in complete medium supplemented with 0.4 mg/mL G418 sulfate. YFP-COG3 stable cell lines were generated as described.38 siRNA-induced knockdowns siRNA duplexes for COG4 and COG728,39 and a set of siRNA to εCOP (siGENOME SMART pool M-017632–02–0005) were obtained from Dharmacon (Chicago, IL). Double transfection was performed on the first and second days using lipofectamine RNAiMAX siRNA Transfection Reagent (Invitrogen) and cells were analyzed 72 h after the second transfection (96 h total). Plasmid preparation and transfection Mammalian expression constructs were generated using standard molecular biology techniques or obtained as generous gifts (Table S1). Plasmids expressing FRB and FKBP12 fragments were obtained from Dr. Gambhir (Stanford University).To
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generate hCOG4-GFP-FRB and hCOG8-GFP-FRB constructs NdeI/KpnI fragments of siRNA-resistant hCOG4 and hCOG834 were sub-cloned into similarly digested c-Luc-FRB plasmid.39 To generate fluorescently-tagged chimeras cDNA encoding full-length proteins were inserted into pECFP-C1, pEGFP-C1 (BspE1/BamHI) or pmCherry-C1-ActA (BspE1/NdeI) vectors. hCOG4–3Myc, hCOG6–3Myc and hCOG8–3Myc were generated as previously described.6,16 hCOG1, hCOG2, mCOG3, hCOG5, and hCOG7 ORFs were inserted into the 3xMyc backbone vector as either HindIII/KpnI (COG1, 5, 7) or NdeI/KpnI (COG2 and 3). hCOG2-Strep-3Myc was made by subcloning the Strep tag40 after the hCOG2 ORF using KpnI site. Multiple expressing plasmids were generated using the multilabel kit from ATG Biosystems according to the manufacturer’s protocol. In brief, COG-3Myc tagged ORFs were inserted into either a donor (ML-DGZ2x and ML-DSZ2cx) or acceptor (ML-AAZ6 and ML-AKZ1) vector as AscI/PacI. The single gene plasmids were then cre-recombined to generate the following two/three gene plasmids: hCOG6–3Myc/hCOG8–3Myc, hCOG5–3Myc/hCOG7–3Myc, and hCOG2-Strep-3Myc/ hCOG4–3Myc/hCOG1–3Myc. Plasmids were isolated from bacterial cells using the QIAprep Spin Miniprep Kit (Qiagen). Plasmid transfections into tissue culture cells were performed with Lipofectamine 2000 (Invitrogen). Immunofluorescence microscopy Cells were plated on glass coverslips one day before transfection. Cells were fixed and stained as described previously.27 Cells were imaged with the 63X oil 1.4 NA objective of a LSM510 Zeiss Laser inverted microscope outfitted with confocal optics. Image acquisition was controlled with LSM510 software (Release Version 4.0 SP1). The “RGB profiler” plug-in of Image J (http:// rsbweb.nih.gov/ij) was used to generate line plots for individual channels. At least three independent experiments were performed to calculate both mean and standard deviation values. SDS–PAGE and western blotting SDS–PAGE and WB were performed as previously described.41 Blots were incubated with primary antibodies, and then with a secondary antibody conjugated with IRDye 680 or IRDye 800 dyes. Blots were scanned and analyzed with an Odyssey Infrared Imaging System (LI-COR). At least three independent experiments were performed to calculate both mean and standard deviation values. Immunoprecipitations Cells were collected and lysed in a 1% Triton X-100 IP buffer (50mM Tris pH 7.4, 150mM NaCl) with 5μL/mL of 100X Halt protease inhibitor cocktail for 30 min on ice. Post-nuclear supernatant was cleared and 90% was added to 20 μL of 50% beads conjugated to llama GFP heavy-chain antibodies (HCAbs) and incubated at 4 °C on a rotator for 2 h. Unbound material was removed, beads were washed three times in 0.05% Triton X-100 in PBS and eluted in 2X sample buffer. FRAP and FLIP experiments HeLa cells that stably express fluorescently tagged proteins were grown on glass-bottom culture plates. FRAP and FLIP were performed using a laser-scanning confocal microscope (LSM 510 Zeiss Laser inverted microscope outfitted with confocal optics)
equipped with an environmental control system (Live cell system, Biovision technologies) set to 37 °C and 5% CO2. Cells were cultured in phenol red–free DMEM/F12 (Sigma-Aldrich) with 1% FBS. In FRAP experiments, prebleached images were taken for 15 s (7 s/frame), and the selected Golgi area was bleached for 15 s using a pulse of the 488-nm laser line at maximal intensity and 100–50 iterations/ROI. After bleaching, fluorescence images were recorded every 15 s for 10 min. In FLIP experiment, a selected area in the cell periphery was bleached every 10 s using a pulse of the 488-nm laser line at maximal intensity and 50 iterations/ROI. Fluorescent images were recorded after each bleaching for 10 min. Signal intensity in the Golgi area was calculated using Zeiss software. Transmission Electron Microscopy Samples were treated according to Valdivia’s lab protocol42 with modifications. In short, cells were fixed for 20 min on ice with 2.5% glutaraldehyde and 0.05% malachite green (EMS) in 0.1M sodium cacodylate buffer, pH 6.8. Samples were post-fixed for 30 min at room temperature with 0.5% osmium tetroxide and 0.8% potassium ferricyanide in 0.1 M sodium cacodylate, incubated for 20 min on ice in 1% tannic acid, and for 1 h in 1% uranyl acetate at room temperature. Specimens were dehydrated with a graded ethanol series, and embedded in Araldite 502/Embed 812 resin (EMS). Ultrathin sections were imaged at 80 kV on a FEI Technai G2 TF20 transmission electron microscope and images were acquired with a FEI Eagle 4kX USB Digital Camera. Knock-sideways assay HeLa cells were grown on coverslips and transfected with siRNA to COG4, COG8 or scrambled siRNA as indicated. Next day cells were transfected with FRB and FKBP12 fusion proteins for 24 h using Lipofectamine 2000 reagent (Invitrogen). Then, after 24 h the cells were treated with 0.2 μM Rapamycin (Cell signaling) in cell culture media for 60 min and were fixed and stained as described previously.27 Cells were imaged with the 63X oil 1.4 NA objective of a LSM510 Zeiss Laser inverted microscope outfitted with confocal optics. Image acquisition was controlled with LSM510 software (Release Version 4.0 SP1). Expression of myc-tagged COG complexes HEK cells were transfected with multi-expression vectors for 36 h using Lipofectamine 2000 (Invitrogen) reagent. (lobe A expression: hCOG1/2/4–3Myc + mCOG3–3Myc; lobe B expression: hCOG6/8–3Myc + hCOG5/7–3Myc; lobe A + lobe B; hCOG1/2/4–3Myc + mCOG3–3Myc + hCOG6/8–3Myc + hCOG5/7–3Myc). After 12 h of COG-Myc expression cells were transfected with GFP expression plasmids using Lipofectamine 2000 (Invitrogen) reagent. Cells were harvested 36 h after initial COG transfection and prepped for IP. Cell fractionation Control and COG KD HeLa cells were washed twice with PBS then treated with a 0.01% saponin solution in buffer A 25 (150mM KCl, 2 mM MgCl2, 20mM Hepes pH7.2, 10% glycerol) for 15 min at 4 °C. After lysis, the soluble material was completely removed and the remaining membrane fraction was collected in buffer A with 1% Triton-X100. Membrane fractionation efficiency was determined by SDS-PAGE. Experiments were performed in triplicates.
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Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed. Acknowledgments
Supplemental Material
We are thankful to Dr. S Gambhir, F Hughson, A Linstedt, S Munro, B Storrie, E Sztul and others who provided reagents and critical reading of the manuscript. We also would like to thank
Supplemental Material may be downloaded here: www. landesbioscience.com/journals/cellularlogistics/article/27888/
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Jacob Szwedo for technical support. This work was supported by the National Institute of Health (GM083144) (VL).
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25. Tebar F, Bohlander SK, Sorkin A. Clathrin assembly lymphoid myeloid leukemia (CALM) protein: localization in endocytic-coated pits, interactions with clathrin, and the impact of overexpression on clathrin-mediated traffic. Mol Biol Cell 1999; 10:2687702; PMID:10436022; http://dx.doi.org/10.1091/ mbc.10.8.2687 26. Suvorova ES, Kurten RC, Lupashin VV. Identification of a human orthologue of Sec34p as a component of the cis-Golgi vesicle tethering machinery. J Biol Chem 2001; 276:22810-8; PMID:11292827; http:// dx.doi.org/10.1074/jbc.M011624200 27. Pokrovskaya ID, Willett R, Smith RD, Morelle W, Kudlyk T, Lupashin VV. Conserved oligomeric Golgi complex specifically regulates the maintenance of Golgi glycosylation machinery. Glycobiology 2011; 21:1554-69; PMID:21421995; http://dx.doi. org/10.1093/glycob/cwr028 28. Shestakova A, Zolov S, Lupashin V. COG complexmediated recycling of Golgi glycosyltransferases is essential for normal protein glycosylation. Traffic 2006; 7:191-204; PMID:16420527; http://dx.doi. org/10.1111/j.1600-0854.2005.00376.x 29. Peanne R, Legrand D, Duvet S, Mir AM, Matthijs G, Rohrer J, Foulquier F. Differential effects of lobe A and lobe B of the Conserved Oligomeric Golgi complex on the stability of beta1,4-galactosyltransferase 1 and alpha2,6-sialyltransferase 1. Glycobiology 2011; 21:864-76; PMID:21062782; http://dx.doi. org/10.1093/glycob/cwq176 30. Reynders E, Foulquier F, Leão Teles E, Quelhas D, Morelle W, Rabouille C, Annaert W, Matthijs G. Golgi function and dysfunction in the first COG4deficient CDG type II patient. Hum Mol Genet 2009; 18:3244-56; PMID:19494034; http://dx.doi. org/10.1093/hmg/ddp262 31. Arasaki K, Takagi D, Furuno A, Sohda M, Misumi Y, Wakana Y, Inoue H, Tagaya M. A new role for RINT-1 in SNARE complex assembly at the trans-Golgi network in coordination with the COG complex. Mol Biol Cell 2013; 24:2907-17; PMID:23885118; http://dx.doi.org/10.1091/mbc.E13-01-0014 32. Kim DW, Sacher M, Scarpa A, Quinn AM, FerroNovick S. High-copy suppressor analysis reveals a physical interaction between Sec34p and Sec35p, a protein implicated in vesicle docking. Mol Biol Cell 1999; 10:3317-29; PMID:10512869; http://dx.doi. org/10.1091/mbc.10.10.3317 33. VanRheenen SM, Cao X, Sapperstein SK, Chiang EC, Lupashin VV, Barlowe C, Waters MG. Sec34p, a protein required for vesicle tethering to the yeast Golgi apparatus, is in a complex with Sec35p. J Cell Biol 1999; 147:729-42; PMID:10562277; http:// dx.doi.org/10.1083/jcb.147.4.729 34. Willett R, Kudlyk T, Pokrovskaya I, Schönherr R, Ungar D, Duden R, Lupashin V. COG complexes form spatial landmarks for distinct SNARE complexes. Nat Commun 2013; 4:1553; PMID:23462996; http://dx.doi.org/10.1038/ncomms2535 35. Andag U, Schmitt HD. Dsl1p, an essential component of the Golgi-endoplasmic reticulum retrieval system in yeast, uses the same sequence motif to interact with different subunits of the COPI vesicle coat. J Biol Chem 2003; 278:51722-34; PMID:14504276; http://dx.doi.org/10.1074/jbc.M308740200
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Cellular Logistics Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/kcll20
Multipronged interaction of the COG complex with intracellular membranes a
a
a
Rose Willett , Irina Pokrovskaya , Tetyana Kudlyk & Vladimir Lupashin a
ab
Department of Physiology and Biophysics; UAMS; Little Rock, AR USA
b
Tomsk State University; Tomsk, Russian Federation Published online: 13 Feb 2014.
Click for updates To cite this article: Rose Willett, Irina Pokrovskaya, Tetyana Kudlyk & Vladimir Lupashin (2014) Multipronged interaction of the COG complex with intracellular membranes, Cellular Logistics, 4:1, e27888, DOI: 10.4161/cl.27888 To link to this article: http://dx.doi.org/10.4161/cl.27888
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Research Paper
Research Paper
Cellular Logistics 4, e27888; February; © 2014 Landes Bioscience
Multipronged interaction of the COG complex with intracellular membranes Rose Willett1, Irina Pokrovskaya1, Tetyana Kudlyk1 and Vladimir Lupashin1,2,* Department of Physiology and Biophysics; UAMS; Little Rock, AR USA; 2Tomsk State University; Tomsk, Russian Federation
1
Keywords: COG complex, Golgi, vesicular trafficking, vesicular tethers, intra-Golgi transport, SNARE, p115, COPI, membrane binding
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Abbreviations: COG, conserved oligomeric Golgi; mCh, mCherry; SNARE, soluble N-ethylmalemide-sensitive fusion attachment protein receptor
The conserved oligomeric Golgi complex is a peripheral membrane protein complex that orchestrates the tethering and fusion of intra-Golgi transport carriers with Golgi membranes. In this study we have investigated the membrane attachment of the COG complex and it’s on/off dynamic on Golgi membranes. Several complimentary approaches including knock-sideways depletion, FRAP, and FLIP revealed that assembled COG complex is not diffusing from Golgi periphery in live HeLa cells. Moreover, COG subunits remained membrane-associated even in COG4 and COG7 depleted cells when Golgi architecture was severely affected. Overexpression of myc-tagged COG sub-complexes revealed that different membrane-associated COG partners including β-COP, p115 and SNARE STX5 preferentially bind to different COG assemblies, indicating that COG subunits interact with Golgi membranes in a multipronged fashion.
Introduction The transport of vesicular carriers in the secretory pathway is facilitated by specialized proteins and protein complexes that assist in formation, movement, tethering, and fusion of trafficking intermediates.1 While several core components of vesicle trafficking machinery including cargo receptors, SNAREs and small GTPases are stably associated with cellular membranes via trans-membrane domains or lipid moieties, other components, including coat proteins, molecular motors and tethering factors, transiently interact with trafficking intermediates and target membranes. In order to achieve both high rate and high specificity of vesicle delivery, membrane attachment of these factors has to be tightly regulated in both a spatial and temporal manner. The conserved oligomeric Golgi (COG) complex belongs to a diverse family of vesicular tethering factors that assist in tethering and fusion of intracellular trafficking intermediates with acceptor membranes in both secretory and endocytic pathways.2-5 The COG complex is comprised of 8 subunits, named COG1–86,7 which are divided into two subcomplexes, lobe A (COG1–4) and lobe B (COG5–8).8,9 Vesicle tethering factors are thought to interact with both vesicular and target membranes through the association with small GTPases, SNAREs, coat proteins, and membrane phospholipids, but in many cases their exact mode of membrane attachment and membrane on-off dynamics remained unknown. Several Golgi tethering factors including giantin and golgin-84 are permanently associated with membranes
via transmembrane domains10,11 while other coiled-coil tethers including p115 rapidly cycle on-off Golgi membrane.12 The membrane association of the subclass of membrane tethers termed the multisubunit tethering complexes (MTC’s),5 of which the COG complex is a member of, is more complicated. The MTC’s are all peripherally associated with membranes through interactions with vesicle targeting/fusion machinery. Intriguingly, the Exocyst complex has been shown to directly interact with Rho GTPases, SNAREs, as well as PI(4,5)P2 through two of its subunits Exo7 and Sec3 to facilitate its membrane attachment.13-15 Likewise with the COG complex, we have demonstrated how interaction of the COG6 subunit with the Golgi SNARE STX6 is critical for COG6 localization on membranes.16 The COG complex is also a known Rab effector, interacting with several different key Golgi Rab proteins including the yeast Ypt1 and Ypt6,17 and the mammalian Rab1a, Rab4a, Rab6a, and Rab30.18 Finally, the COG complex will also interact with βCOP and coiled-coil tethers p115, GM130, Giantin, golgin84, TMF, and CASP.18-21 This extensive range of COG interactions with trafficking regulators provides for a multipronged method of attachment of COG subunits to Golgi and vesicle membranes. In this study we have investigated the membrane attachment of the COG complex and it’s on/off dynamic on Golgi membranes. We demonstrate that while individual COG subunits are capable of freely diffusing in a cell, once in their complex/ subcomplex, the subunits will not readily move from Golgi membranes. Next, we unveil the tendency for individual COG
*Correspondence to: Vladimir Lupashin; Email: [email protected] Submitted: 11/25/13; Accepted: 01/16/14 Citation: Willett R, Pokrovskaya I, Kudlyk T, Lupashin V. Multipronged interaction of the COG complex with intracellular membranes. Cellular Logistics 2014; 4:e27888; http://dx.doi.org/10.4161/cl.27888 www.landesbioscience.com Cellular Logistics e27888-1
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Figure 1. For figure legend, see page 3.
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Figure 1 (see previous page). Transiently expressed COG subunits are efficiently relocalized to mitochondria in a knock-sideways assay. A schematic diagram of the mitochondrial knock-sideways protein constructs (A). HeLa cells were transiently transfected with FKBP12-mCherry-ActA and GFP-FRB (B, C), COG4-GFP-FRB (D, E), or COG8-GFP-FRB (F, G). 24hrs after transfection cells were treated with 0.2μM rapamycin for 60 min. After rapamycin treatment cells were fixed and analyzed by confocal microscopy. Line plots for overlap between red and green channels are shown measuring the relative value of signal intensity (y-axis) over the distance measured in pixels (x-axis). Size bar, 10 μm.
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subunits to remain bound to Golgi membranes in a fractionation assay, despite the disturbance of the COG complex via siRNA COG depletion. Finally, we reveal a preference for COG complex/subcomplex interactions with COG interacting trafficking regulators using a complete COG complex expression model. These results demonstrate that the attachment of the COG complex to Golgi membranes is multipronged and facilitated through multiple interactions.
Results The COG complex is tightly associated with Golgi membranes To investigate the dynamics of individual COG subunits on the Golgi membrane we first applied a “knock-sideways” strategy of rapamycin-inducible capturing of soluble proteins on mitochondria membranes22. Plasmids encoding mitochondriatargeted FKBP12-mCherry-ActA as well as siRNA-resistant COG4 and COG8 with attached GFP-FRB tag were constructed (Fig. 1A). Soluble GFP-FRB was used as a positive control. Transient (less than 30 h) expression of GFP-FRB-tagged proteins revealed that, upon expression in HeLa cells, the majority of expressed proteins demonstrate a diffuse pattern, indicating soluble cytoplasmic localization (Fig. 1B, D and F). This phenotype likely indicates a delay in the incorporation of tagged COG subunits into functional Golgi-localized complexes due to a competition with the endogenous COG proteins. Indeed, longer expression demonstrated partial localization of tagged COG subunits on the Golgi membrane (data not shown). As expected, the FKBP12-mCherry-ActA construct was extensively localized to the mitochondria and addition of rapamycin to cells induced rapid and specific relocalization of all GFP-FRB tagged soluble protein to the mitochondrial vicinity (Fig. 1C, E and G). This data indicates that soluble COG subunits are freely diffusible in HeLa cells. We next wanted to investigate the dynamics of COG subunits in the assembled COG complex. Using a gene replacement strategy, we first knocked-down the endogenous COG4 and COG8 with the corresponding siRNA, and then transfected cells with plasmids expressing siRNA-resistant GFP-FRB-tagged proteins. As expected, this strategy allowed for the efficient incorporation of GFP-FRB –tagged proteins into Golgi-localized COG complexes (Fig. 2A and C). Surprisingly, upon rapamycin treatment, the assembled COG subunits failed to relocalize to FKBP12mCherry-ActA-decorated mitochondria, indicating that the COG complex is not diffusing freely from the Golgi vicinity (Fig. 2B and D). This was also evident after longer treatment, with 2, 4, and 24 h incubation with Rapamycin still unable to relocalize the GFP-FRB tagged COG subunits to the mitochondria (data not shown).
The assembled COG complex has a molecular weight ~700 KD7,17,23 and therefore may diffuse in the cytoplasm with much slower dynamics as compared with individual COG subunits. To test this hypothesis we wanted to compare the COG dynamic with that of COPI vesicle coat, a protein complex that has a similar size and is rapidly assembling/disassembling during the vesicular trafficking cycle24. siRNA-resistant GFP-FRB-tagged εCOP was properly localized in HeLa cells (Fig. 2D) and rapidly relocalized to the mitochondria decorated with FKBP12-mCherryActA (Fig. 2F), indicating that the COG complex’s size does not play a significant role in this relocalization assay. This reinforced the notion that the assembled COG complex is not diffusing away from the Golgi vicinity. We next wanted to explore the dynamic of the COG subunits in an in vivo setting. To achieve this, we performed FRAP (fluorescence recovery after photobleaching) (Fig. 3A) and FLIP (fluorescence loss in photobleaching) (Fig. 3B) assays in cells stably expressing YFP-COG3 or COG8-GFP-FRB. For both COG3 and COG8, the t1/2 for maximum recovery of fluorescence was significantly increased compared with the v-SNARE GFP-GS15 and the small GTPase YFP-Arf1 (Fig. 3A and C). This slow recovery is indicative of a lack of diffusible COG complex in the cell, and suggests that the majority of COG subunits constantly remaining in the Golgi vicinity. Moreover, the FRAP of YFP-hCOG3 had a slower recovery than hCOG8-GFP-FRB (t1/2 of 152.6s and 122s respectively) indicating that lobe A subunits are potentially more tightly retained on membranes than lobe B subunits. In line with this data, we saw that in the FLIP experiments that the loss of COG8-GFP-FRB was similar to that of v-SNARE GFP-GS15 and much slower than the YFP-Arf1, suggesting the tight association of the COG complex with Golgi membranes and/or the Golgi vicinity. COG subunit localization in COG compromised cells Having demonstrated how COG subunits once in their complex remain tightly associated with Golgi vicinity, we next wanted to explore their membrane association when the complex is partially disrupted. Using a saponin fractionation assay25 we separated WT and COG4 depleted HeLa cells into membrane and cytosol fractions, and then determined the membrane association of COG subunits 3, 4, 6, and 8. In WT cells the distribution of the COG subunits in membrane and cytosol fractions is 40%:60%, respectively (Fig. 4A and B). This is in good agreement with COG fractionation data obtained from mechanically disrupted HeLa cells26. Upon depletion of COG4, the saponinextractible pool of COG subunits was increased, but surprisingly a significant fraction (20–30%) of both lobe A subunit COG3 and lobe B subunits COG6 and COG8 was still associated with saponin-resistant membranes. Confocal microscopy revealed that COG3 remained associated with Golgi membranes in both WT and COG4-depleted cells while COG8 changed its localization
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Figure 2. COG subunits that assemble on the Golgi membrane are not available for the mitochondria capturing. HeLa cells were treated with COG4 siRNA (A, B), COG8 siRNA (C, D) or εCOP siRNA (E, F). Twenty-four hours after siRNA treatment cells were transfected with FKBP12-mCherry-ActA and COG4-GFP-FRB (A, B), COG8-GFP-FRB (C, D), or εCOP (E, F). 48 h after second transfection cells were treated with 0.2μM rapamycin for 60 min, fixed and analyzed by confocal microscopy. Line plots for overlap between red and green channels are shown measuring the relative value of signal intensity (y-axis) over the distance measured in pixels (x-axis). Size bar, 10 μm.
from the Golgi in WT cells, to a dispersed puncta-like pattern in COG4-depleted cells (Fig. 4C and D). These puncta are likely to
represent COG8’s association with Golgi-derived vesicles which are dramatically accumulated in cells upon COG4 depletion
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(compare Golgi area in electron micrograph in Fig. 4E and F, arrows). We, and others, have demonstrated previously that depletion of COG4 and COG7 in HeLa cells have the most dramatic effect on Golgi morphology and stability of Golgi-localized components of glycosylation machinery.27-29 Evaluation of COG subunit localization in COG7depleted cells revealed that the membrane association of lobe A subunits was slightly increased (Fig. 5A and B). The level of COG6 was significantly decreased in COG7-depleted cells, while the level of another lobe B subunit COG8 was not affected and remained mostly associated with the membrane fraction. Again, like in the case of COG4-depleted cells, COG8 signal was primarily found in dispersed puncta (Fig. 5C) and not associated with severely dilated and fragmented (Fig. 5D) Golgi membranes. This data indicates that even a partially destroyed COG complex, and/or remaining sub-complexes, maintain their membrane association. COG subcomplexes selectively interact with membrane bound partners To get insights into the molecular nature of the interaction of COG subcomplexes with their membrane partners, we have co- expressed preassembled lobe A, lobe B, or the complete COG complex with GFP-tagged partner proteins. In this set-up every COG subunit was labeled with the same 3xmyc tag allowing for precise quantification of COG-partner interactions. Our analysis revealed that individual lobe A and lobe B subcomplexes were capable of interaction with the COPI coat complex via the C-terminus of the βCOP subunit,18 whereas interaction of the completely assembled COG complex was significantly lower (Fig. 6A). In contrast, the coiled-coil tether p115 preferably interacted with lobe A, as well as the whole COG complex (Fig. 6B), while Golgi SNARE STX5 was preferentially binding to lobe B and the whole COG complex (Fig. 6C). This data indicates that different COG assemblies
Figure 3. Lobe A and lobe B COG subunits display different kinetic mobility profiles. HeLa cells stably expressing YFP-Arf1, GFP-GS15, YFP-hCOG3, or hCOG8-GFP-FRB were incubated at 37 °C and visualized with a LSM 510 laser confocal microscope. (A) Averaged FRAP experiments bleaching an ROI of the Golgi for 15s then measuring the recovery every 15s for 10min. (B) Averaged FLIP experiments bleaching an area in the cell periphery every 15s for 10min and measuring the signal loss for the Golgi ROI.. (C) FRAP t1/2 values. n denotes number of cells quantified. ROI, regions of interest.
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Figure 4. COG lobe B subunits lose Golgi localization in in COG4 KD cells, but remained membrane-associated. Control and 96h COG4 KD HeLa cells were treated with 0.01% saponin to fractionate into membrane and cytosol fractions (A). Fractions were separated on an SDS–PAGE gel, transferred to nitrocellulose membrane, and probed with antibodies against COG3, COG4, COG6, and COG8. Membrane and cytosol separation was measured by blotting against cytosolic GAPDH and membrane associated Vti1a proteins. Experiments were performed in triplicates. (B) Quantification of the COG subunits in the membrane associated fraction. HeLa T2-GFP control (C) and 96h COG4 KD (D) cells plated on coverslips were fixed and stained with COG3 and COG8 antibodies and analyzed by confocal microscopy. Electron micrograph of control (E) and 96h COG4 KD (F) HeLa cells fixed and processed for TEM. Arrows denote Golgi membranes, “M” denotes mitochondria. * denotes non-specific band recognized by COG6 antibodies. Error bars denote standard deviation. Size bar, 10 μm unless otherwise noted.
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Figure 5. COG subunits remain membrane associated in cells depleted for COG7. Control and 96h COG7 KD HeLa cells were treated with 0.01% saponin and fractionated into membrane and cytosol fractions (A). Fractions were separated on an SDS–PAGE gel, transferred to nitrocellulose membrane, and probed with antibodies against COG3, COG4, COG6, and COG8. Membrane and cytosol separation was measured by blotting against cytosolic GAPDH and membrane associated Vti1a. Experiments were performed in triplicates. (B) Quantification of the COG subunits in the membrane associated fraction. HeLa T2-GFP 96h COG7 KD (C) cells plated on coverslips were fixed and stained with COG3 and COG8 antibodies and analyzed by confocal microscopy. Electron micrograph 96h COG7 KD (D) HeLa cells fixed and processed for TEM. Arrows denote Golgi membranes, “M” denotes mitochondria. Error bars denote standard deviation. Size bar, 10 μm, unless otherwise noted.
possess different specificity for their membrane-bound partners. It is this multipronged interaction profile that is likely to be responsible for the tight association of the COG complex with the Golgi membranes.
Discussion In this study we illustrated how the COG complex is tightly associated with Golgi vicinity through multiple interactions with trafficking regulating components. Using a
knock-sideways approach we revealed that soluble expressed COG subunits are capable of being mis-localized to non secretory compartments, indicating that they are freely diffusible. This is in stark contrast to COG subunits once incorporated into the COG complex, and thus membrane localized, which are no longer diffusing throughout the cell. This lack of diffusion on and off of Golgi vicinity is also characterized by the strong tendency of COG subunits to remain membrane bound despite disturbances of the COG complex via depletion of individual subunits.
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Figure 6. COG subcomplexes show binding preference to different membrane-associated partners. HEK 293 cells transiently transfected with COGMyc multi-expression plasmids and GFP tagged partner proteins as indicated (lobe A expression: hCOG1/2/4–3Myc + mCOG3–3Myc; lobe B expression: hCOG6/8–3Myc + hCOG5/7–3Myc; lobe A + lobe B; hCOG1/2/4–3Myc + mCOG3–3Myc + hCOG6/8–3Myc + hCOG5/7–3Myc). (A) GFP-βCOP-CTD (aa 666–953) (B) GFP-P115 (C) GFP-STX5, and (E) GFP as a negative control for COG-Myc binding. 36 h post transfection, cells were collected and lysed, and GFP–SNARE-interacting proteins were precipitated with anti-GFP antibodies. Immuno-precipitates, along with 5% or 10% of total input, were separated on an SDS–PAGE gel, transferred to nitrocellulose membrane and probed with antibodies against Myc (upper panel) and GFP (lower panel). (F) Quantification of COG-Myc binding.
This tight membrane association of COG subunits to Golgi membranes despite depletions of individual subunits is not a new concept. We have shown that in yeast, individual COG subunit depletion still results in all subunits present in a total membrane fraction8. Additionally, primary fibroblasts from a
COG4 mutation CDG-II patient revealed that despite a lack of membrane association of the R729W COG4 point mutant, both lobe A and lobe B COG subunits still remain membrane bound.30 These results are further supportive of our conclusion that the membrane attachment of the COG complex to Golgi/
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vesicle membranes is achieved through multiple interactions with individual COG subunits. We demonstrate for the first time an expression model wherein all 8 COG subunits are expressed and pre-assembled to characterize COG interactions at a physiological and quantifiable setting. Gel filtration of HeLa cell membrane and cytosol fractions has eluded to a lack of monomeric COG subunits at steady-state (Willett and Lupashin, in progress), indicating that COG subunits are always in either a complex or subcomplex in the cell. In light of this, we developed this assay to now probe for COG interactions in their native conformations allowing for a more concise interpretation of the COG interactome.4 Indeed, we see that the expression of subcomplexes/complex adds an additional level of selectivity of COG interactions to highlight those which are more physiological. This exciting new assay urges for a confirmation of many of the individual protein-protein interactions that have been previously described for the COG complex.16,18-20,31 These results also shed new light on the steady-state membrane/ cytosol distribution of the COG complex. Previously it was published that the yeast COG complex was preferentially in a soluble pool32. This split was identified using spheroplasts that underwent harsh conditions (NaN3 pre-treatments). Alternatively, mechanically disrupted yeast and mammalian cells demonstrated preferentially membrane localization of COG subunits.26,33 In this work we have seen that the split between the membrane bound and soluble pool of the COG complex using a gentle saponin extraction protocol is closer to a 40:60 split (Fig. 4A and B). This 40:60 split may also be an over-statement of the soluble pool of the COG complex as seen with FRAP and FLIP assays (Fig. 3). In a live cell situation, we see that there is very little recovery of fluorescence after bleaching, indicating that there is not a readily available diffusible pool of COG complex, and rather that the majority of the protein is always present in the Golgi vicinity. We hypothesize that the appearance of the soluble pool in fractionation studies is due to the rapid loss of association of the COG complex with Golgi membranes upon disruption of cells. In order for the COG complex to be functional in tethering retrograde vesicles at the Golgi, it must first and foremost be properly localized to Golgi area. Our previous work has shown that one of the functions of the COG complex is to serve as a landmark for two SNARE complexes, the cis-Golgi localized STX5 containing SNARE complex, and the trans-Golgi localized STX16 containing SNARE complex.34 In order for the COG complex to organize these SNARE complexes, it is critical that they interact in the correct Golgi compartment. We believe that it is through the COGs multiple interaction profiles with membrane-bound partners (i.e., Rabs, SNAREs, coiled-coil tethers such as p115) that the compartment specificity of the COG complex is achieved. The interaction detected between the COPI coatomer and the COG subcomplexes also contributes to an interesting model of COG mediated vesicle fusion. As indicated, the βCOP subunit is interacting almost exclusively with the lobe A and lobe B subcomplexes (Fig. 6A). Therefore, we can envision a model wherein COPI coat (either on intra-Golgi vesicle or bound onto Golgi membranes) is destabilized upon interaction with COG subunits, thus uncoating the membrane and allowing for fusion. Support
for this hypothesis is found with another multisubunit tethering complex, the Dsl1 complex, which has been shown to directly interact with COPI subunits α-COP and δ-COP via Dsl1p35-37 and depletion of this Dsl1 complex subunit leads to an accumulation of COPI coated vesicles. Currently it is still unknown whether or not the COG complex has the same ability to uncoat vesicles and is a topic that requires further investigation. Given the low overall co-IP efficiencies we detect in our assays, we can also conclude that COG-membrane partner interactions are very transient and unstable in an in vitro setting. This multipronged interaction approach still raises many questions, including the time relationship of the interactions. It will be very important to know whether these interactions are occurring simultaneously or if there is a hand-off of the COG complex from the different interacting partners.
Methods Reagents and antibodies Protein G agarose was purchased from Roche. Antibodies used for immunofluorescence (IF) microscopy or western blotting (WB) were purchased through commercial sources, received as gifts from generous individual investigators, or generated by us via affinity purification. Antibodies were as follows: Rabbit polyclonal: Myc (Bethyl Laboratories); COG3, COG4, COG6, COG8 (this lab).27 Mouse monoclonal: GFP (Molecular Probes); GFP (Covance); GAPDH (Santa Cruz); Myc (Cell Signaling); Vti1a (BD Biosciences). Secondary IRDye 680 goat anti-rabbit, IRDye 700 goat anti-mouse and IRDye 800 donkey anti-goat for WB were from LI-COR Biosciences. Anti-rabbit HiLyte 488, HiLyte 555, and DyLight647 for IF were obtained from AnaSpec and Jackson ImmunoResearch, Inc. Cell culture HeLa cells were cultured in DMEM/F-12 medium (Thermo Scientific) supplemented with 15 mM HEPES, 2.5 mM L-glutamine and 10% FBS (Atlas Biologicals) at 37 °C and 5% CO2 in a 90% humidified incubator. GFP-GS15, YFP-Arf1 and COG8-GFP-FRB stable cell lines were generated by transfecting cells with corresponding plasmids (Table S1) followed by selection for G418 resistance in complete medium supplemented with 0.4 mg/mL G418 sulfate. YFP-COG3 stable cell lines were generated as described.38 siRNA-induced knockdowns siRNA duplexes for COG4 and COG728,39 and a set of siRNA to εCOP (siGENOME SMART pool M-017632–02–0005) were obtained from Dharmacon (Chicago, IL). Double transfection was performed on the first and second days using lipofectamine RNAiMAX siRNA Transfection Reagent (Invitrogen) and cells were analyzed 72 h after the second transfection (96 h total). Plasmid preparation and transfection Mammalian expression constructs were generated using standard molecular biology techniques or obtained as generous gifts (Table S1). Plasmids expressing FRB and FKBP12 fragments were obtained from Dr. Gambhir (Stanford University).To
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generate hCOG4-GFP-FRB and hCOG8-GFP-FRB constructs NdeI/KpnI fragments of siRNA-resistant hCOG4 and hCOG834 were sub-cloned into similarly digested c-Luc-FRB plasmid.39 To generate fluorescently-tagged chimeras cDNA encoding full-length proteins were inserted into pECFP-C1, pEGFP-C1 (BspE1/BamHI) or pmCherry-C1-ActA (BspE1/NdeI) vectors. hCOG4–3Myc, hCOG6–3Myc and hCOG8–3Myc were generated as previously described.6,16 hCOG1, hCOG2, mCOG3, hCOG5, and hCOG7 ORFs were inserted into the 3xMyc backbone vector as either HindIII/KpnI (COG1, 5, 7) or NdeI/KpnI (COG2 and 3). hCOG2-Strep-3Myc was made by subcloning the Strep tag40 after the hCOG2 ORF using KpnI site. Multiple expressing plasmids were generated using the multilabel kit from ATG Biosystems according to the manufacturer’s protocol. In brief, COG-3Myc tagged ORFs were inserted into either a donor (ML-DGZ2x and ML-DSZ2cx) or acceptor (ML-AAZ6 and ML-AKZ1) vector as AscI/PacI. The single gene plasmids were then cre-recombined to generate the following two/three gene plasmids: hCOG6–3Myc/hCOG8–3Myc, hCOG5–3Myc/hCOG7–3Myc, and hCOG2-Strep-3Myc/ hCOG4–3Myc/hCOG1–3Myc. Plasmids were isolated from bacterial cells using the QIAprep Spin Miniprep Kit (Qiagen). Plasmid transfections into tissue culture cells were performed with Lipofectamine 2000 (Invitrogen). Immunofluorescence microscopy Cells were plated on glass coverslips one day before transfection. Cells were fixed and stained as described previously.27 Cells were imaged with the 63X oil 1.4 NA objective of a LSM510 Zeiss Laser inverted microscope outfitted with confocal optics. Image acquisition was controlled with LSM510 software (Release Version 4.0 SP1). The “RGB profiler” plug-in of Image J (http:// rsbweb.nih.gov/ij) was used to generate line plots for individual channels. At least three independent experiments were performed to calculate both mean and standard deviation values. SDS–PAGE and western blotting SDS–PAGE and WB were performed as previously described.41 Blots were incubated with primary antibodies, and then with a secondary antibody conjugated with IRDye 680 or IRDye 800 dyes. Blots were scanned and analyzed with an Odyssey Infrared Imaging System (LI-COR). At least three independent experiments were performed to calculate both mean and standard deviation values. Immunoprecipitations Cells were collected and lysed in a 1% Triton X-100 IP buffer (50mM Tris pH 7.4, 150mM NaCl) with 5μL/mL of 100X Halt protease inhibitor cocktail for 30 min on ice. Post-nuclear supernatant was cleared and 90% was added to 20 μL of 50% beads conjugated to llama GFP heavy-chain antibodies (HCAbs) and incubated at 4 °C on a rotator for 2 h. Unbound material was removed, beads were washed three times in 0.05% Triton X-100 in PBS and eluted in 2X sample buffer. FRAP and FLIP experiments HeLa cells that stably express fluorescently tagged proteins were grown on glass-bottom culture plates. FRAP and FLIP were performed using a laser-scanning confocal microscope (LSM 510 Zeiss Laser inverted microscope outfitted with confocal optics)
equipped with an environmental control system (Live cell system, Biovision technologies) set to 37 °C and 5% CO2. Cells were cultured in phenol red–free DMEM/F12 (Sigma-Aldrich) with 1% FBS. In FRAP experiments, prebleached images were taken for 15 s (7 s/frame), and the selected Golgi area was bleached for 15 s using a pulse of the 488-nm laser line at maximal intensity and 100–50 iterations/ROI. After bleaching, fluorescence images were recorded every 15 s for 10 min. In FLIP experiment, a selected area in the cell periphery was bleached every 10 s using a pulse of the 488-nm laser line at maximal intensity and 50 iterations/ROI. Fluorescent images were recorded after each bleaching for 10 min. Signal intensity in the Golgi area was calculated using Zeiss software. Transmission Electron Microscopy Samples were treated according to Valdivia’s lab protocol42 with modifications. In short, cells were fixed for 20 min on ice with 2.5% glutaraldehyde and 0.05% malachite green (EMS) in 0.1M sodium cacodylate buffer, pH 6.8. Samples were post-fixed for 30 min at room temperature with 0.5% osmium tetroxide and 0.8% potassium ferricyanide in 0.1 M sodium cacodylate, incubated for 20 min on ice in 1% tannic acid, and for 1 h in 1% uranyl acetate at room temperature. Specimens were dehydrated with a graded ethanol series, and embedded in Araldite 502/Embed 812 resin (EMS). Ultrathin sections were imaged at 80 kV on a FEI Technai G2 TF20 transmission electron microscope and images were acquired with a FEI Eagle 4kX USB Digital Camera. Knock-sideways assay HeLa cells were grown on coverslips and transfected with siRNA to COG4, COG8 or scrambled siRNA as indicated. Next day cells were transfected with FRB and FKBP12 fusion proteins for 24 h using Lipofectamine 2000 reagent (Invitrogen). Then, after 24 h the cells were treated with 0.2 μM Rapamycin (Cell signaling) in cell culture media for 60 min and were fixed and stained as described previously.27 Cells were imaged with the 63X oil 1.4 NA objective of a LSM510 Zeiss Laser inverted microscope outfitted with confocal optics. Image acquisition was controlled with LSM510 software (Release Version 4.0 SP1). Expression of myc-tagged COG complexes HEK cells were transfected with multi-expression vectors for 36 h using Lipofectamine 2000 (Invitrogen) reagent. (lobe A expression: hCOG1/2/4–3Myc + mCOG3–3Myc; lobe B expression: hCOG6/8–3Myc + hCOG5/7–3Myc; lobe A + lobe B; hCOG1/2/4–3Myc + mCOG3–3Myc + hCOG6/8–3Myc + hCOG5/7–3Myc). After 12 h of COG-Myc expression cells were transfected with GFP expression plasmids using Lipofectamine 2000 (Invitrogen) reagent. Cells were harvested 36 h after initial COG transfection and prepped for IP. Cell fractionation Control and COG KD HeLa cells were washed twice with PBS then treated with a 0.01% saponin solution in buffer A 25 (150mM KCl, 2 mM MgCl2, 20mM Hepes pH7.2, 10% glycerol) for 15 min at 4 °C. After lysis, the soluble material was completely removed and the remaining membrane fraction was collected in buffer A with 1% Triton-X100. Membrane fractionation efficiency was determined by SDS-PAGE. Experiments were performed in triplicates.
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Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed. Acknowledgments
Supplemental Material
We are thankful to Dr. S Gambhir, F Hughson, A Linstedt, S Munro, B Storrie, E Sztul and others who provided reagents and critical reading of the manuscript. We also would like to thank
Supplemental Material may be downloaded here: www. landesbioscience.com/journals/cellularlogistics/article/27888/
References
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Jacob Szwedo for technical support. This work was supported by the National Institute of Health (GM083144) (VL).
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