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© 2011 John Wiley & Sons A/S doi:10.1111/j.1600-0854.2011.01257.x
Differential Selection of Golgi Proteins by COPII Sec24 Isoforms in Procyclic Trypanosoma brucei Lars Demmel1 , Michael Melak1 , Harald Kotisch1 , Justin Fendos2 , Siegfried Reipert1 and Graham Warren1,∗ 1 Max
F. Perutz Laboratories, University of Vienna, Medical University of Vienna, Dr. Bohrgasse 9, A-1030 Vienna, Austria 2 Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511, USA *Corresponding author: Graham Warren,
[email protected] The Sec24 subunit of the coat protein complex II (COPII) has been implicated in selecting newly synthesized cargo from the endoplasmic reticulum (ER) for delivery to the Golgi. The protozoan parasite, Trypanosoma brucei, contains two paralogs, TbSec24.1 and TbSec24.2, which were depleted using RNA interference in the insect form of the parasite. Depletion of either TbSec24.1 or TbSec24.2 resulted in growth arrest and modest inhibition of anterograde transport of the putative Golgi enzyme, TbGntB, and the secretory marker, BiPNAVRG-HA9. In contrast, depletion of TbSec24.1, but not TbSec24.2, led to reversible mislocalization of the Golgi stack proteins, TbGRASP and TbGolgin63. The latter accumulated in the ER. The localization of the COPI coatomer subunit, TbεCOP, and the trans Golgi network (TGN) protein, TbGRIP70, was largely unaffected, although the latter was preferentially lost from those Golgi that were not associated with the bilobe, a structure previously implicated in Golgi biogenesis. Together, these data suggest that TbSec24 paralogs can differentiate among proteins destined for the Golgi. Key words: COPII vesicles, ER, ERES, Golgi, membrane trafficking, Sec24 Received 28 January 2011, revised and accepted for publication 26 July 2011, uncorrected manuscript published online 29 July 2011, published online 6 September 2011
Secretory proteins or proteins destined for delivery to the endosomal/lysosomal system are synthesized and assembled in the endoplasmic reticulum (ER), transported first to the Golgi and then to their final destination (1,2). Most, if not all, of these proteins leave the ER in coat protein complex II (COPII)-coated vesicles (3), which separate them away from ER-resident proteins and deliver them to the cis, or entry face, of the Golgi. COPII vesicles are assembled in a step-wise manner. The first event in COPII vesicle formation is the recruitment of Sar1 GTPase to the cytoplasmic face of the ER. At the ER, the guanine nucleotide exchange factor, Sec12, catalyzes
the GDP for GTP exchange on Sar1 leading to its activation and exposure of an amphiphatic α-helix, which is inserted into the ER membrane (4–8). Membrane-bound Sar1 then specifically interacts with Sec23 resulting in the recruitment of the heterodimeric Sec23/Sec24 complex (9,10). By binding to ER export motifs within the cytoplasmic domain of membrane-bound cargo proteins or to receptors for luminal soluble proteins, Sec24 recruits cargo proteins into the forming vesicle (11–15). Subsequently, the COPII outer layer, comprising Sec13/Sec31 heterotetramers, binds to and concentrates the Sar1/Sec23/Sec24 prebudding complexes (16), thereby enhancing membrane deformation and leading, eventually, to scission and release of a COPII vesicle. The Sec23 component acts as a GTPase-activating protein to stimulate the Sar1 GTPase activity, triggering uncoating and recycling of coat components for further rounds of vesicle budding (17–19). The Sec24 subunit is responsible for packaging specific cargo into COPII vesicles and most organisms have more than one isoform (13,14,20–22). Budding yeast contains three paralogs (Sec24p, Lst1p and Iss1p) of which only Sec24p is essential for growth, whereas mammalian cells (in culture) have four non-essential isoforms (Sec24A–D) (23). In terms of cargo selection, these isoforms can sometimes complement each other. Overexpression of the nonessential and low abundance yeast Sec24 isoform, Iss1p, can rescue the growth of sec24ts cells at the restrictive temperature. In addition, the major plasma membrane ATPase, Pma1p, requires Sec24p for transport to the cell surface, but the presence of another isoform, Lst1p, increases its efficiency (24–26). Sec24p has a mutually exclusive binding site for either the trans Golgi network (TGN) membrane protein, Sys1p, or the SNARE, Bet1p (14). Mutants of Sec24p that can no longer bind to Sys1p or Bet1p cannot support growth unless either Iss1p and/or Lst1p are present (14). This is despite the fact that Lst1p cannot package Sys1p or Bet1p into COPII vesicles in vitro, although the binding region is well conserved between the two Sec24p isoforms (13,14). Together, these data suggest that different Sec24 isoforms can have overlapping specificity. In mammalian cells, Sec24A is preferentially used for the ER exit of cargo with aromatic/hydrophobic signal motifs. However, the remaining isoforms in combination are able to compensate for the depletion of Sec24A (27). The different Sec24 isoforms are not always capable of complementation. Vangl2, a component of the Wnt signaling pathway, is exclusively sorted into COPII vesicles by Sec24B in mice in vivo. Mice harboring non-functional www.traffic.dk 1575
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alleles of Sec24B fail to close their neural tube and die during development (28,29). It has further been shown that some SNARE proteins involved in the ER-to-Golgi transport are bound and packaged into COPII vesicles by a specific subset of Sec24 isoforms in yeast and mammals (13,14,21,22). Glycosylphosphatidylinositol (GPI)-anchored proteins are another example of cargo carried by COPII vesicles. The yeast GPI-anchored protein, Gas1p, is sorted and packaged into COPII vesicles using Emp24p, a member of the p24 cargo receptor family (30–32). The p24 proteins are highly conserved membrane-spanning proteins that can act as adaptors, binding COPII components with their cytoplasmic domains and soluble cargo proteins and GPI-anchored proteins with their luminal receptor domains. Deletion of the Sec24 paralog Lst1p delays the surface delivery of Gas1p (25) and in vitro reconstitution suggests that Gas1p exits the ER in distinct COPII vesicles that are devoid of other secreted proteins (33). In addition, the mammalian GPI-anchored protein CD59 preferentially uses Sec24C and Sec24D for exit from the ER (34). GPI-anchored proteins were first identified (35) and extensively studied in the protist parasite Trypanosoma brucei, the causative agent of African trypanosomiasis in humans and Nagana in cattle. The parasite exists in two life cycle stages, the bloodstream form (BSF) inhabiting the vertebrate host and the procyclic (insect) form (PCF) in the Tsetse fly. For BSFs, the primary mechanism for evading the vertebrate immune response is a dense surface layer of variable surface glycoprotein (VSG), a GPI-anchored protein. Trypanosomes express only one antigenically distinct copy of VSG at any one time and there are 107 molecules/cell that comprise more than 98% of the cell surface proteins.
Trypanosoma brucei contains two paralogs of Sec24 as well as Sec13 and Sec23. TbSec24 and TbSec23 form obligate pairs: TbSec24.1/TbSec23.2 and TbSec24.2/TbSec 23.1. Interestingly, the former pair preferentially transports VSG coat protein, in particular, and GPI-anchored proteins, in general, in the BSF of the parasite. By way of contrast, the lysosomal proteins TbbCATL and p67 exhibit no preference for either TbSec24 isoform (36).
Results Localization of TbSec24.1 and TbSec24.2 isoforms in procyclic T. brucei cells To investigate the localization of the inner COPII subunits TbSec24 and to determine their endogenous protein levels in procyclic T. brucei cells, polyclonal antibodies were raised against two peptides within both TbSec24.1 and TbSec24.2. The peptides were chosen for their antigenicity and their low sequence identity between the two TbSec24 isoforms. Immunoblots of T. brucei cell lysates using affinity-purified anti-TbSec24.1 or anti-TbSec24.2 antibodies showed single protein bands, which matched the predicted molecular weights of 108.1 and 100.8 kDa, respectively (Figure 1A). The antibodies were used for immunofluorescence microscopy and labeled a structure between the kinetoplast (containing all mitochondrial DNA) and the nucleus, the known location of the ER exit site (ERES) by electron microscopy (EM) (39,40) (Figure 1B). Endogenous replacement cell lines, in which the viral Ty epitope tag was fused to the N-terminus of TbSec24.1 or TbSec24.2, were created to perform colocalization experiments with established antibodies against organelle markers. Ty antibodies detected Ty-TbSec24.1 and Ty-TbSec24.2 as single protein bands of the expected sizes in immunoblots of total cell lysates (data not shown). To confirm the localization of TbSec24.1 and TbSec24.2 to the ERES, cell lines expressing Ty-TbSec24.1 and Ty-TbSec24.2 were stably transfected with yellow fluorescent protein (YFP)-tagged TbSec13.1, which has previously been shown by immuno-EM to localize to the ERES in T. brucei (38). Ty-TbSec24.1 and Ty-TbSec24.2 localization by immunofluorescence microscopy of methanolfixed cells recapitulated the localization pattern observed using TbSec24.1 and TbSec24.2 antibodies (Figure 1B). In agreement with the data obtained in other organisms, showing that the inner and outer layer COPII components colocalize (41–43), YFP-TbSec13.1 extensively colocalized with both Ty-TbSec24.1 and Ty-TbSec24.2 (Figure 2A).
We wondered whether there might also be selective delivery to other destinations along the secretory pathway. We are interested in the biogenesis of the Golgi and we have been using T. brucei as a model system particularly since it has a single Golgi that undergoes duplication during the cell cycle (37,38). We wondered whether the different components needed to build a Golgi might be packaged into COPII vesicles by binding specifically to different TbSec24 isoforms.
Next, the localization of TbSec24 isoforms relative to the Golgi and the TGN was examined. Methanol-fixed cells expressing Ty-TbSec24.1 or Ty-TbSec24.2 were labeled with antibodies against the Golgi stack marker TbGRASP (Golgi r eassembly stacking protein, Tb11.02.0260) (38) or the TGN marker TbGRIP70 (GRIP domain containing protein with molecular weight of 70 kDa, Tb11.02.5040) (44). As expected, Ty-TbSec24.1 and Ty-TbSec24.2 were adjacent to Golgi labeled with TbGRASP and TbGRIP70. The Ty-TbSec24 isoforms were more tightly juxtaposed with TbGRASP than TbGRIP70 (Figure 2B,C). This supports the canonical hierarchy of the ERES (TbSec24, TbSec13.1), the Golgi stack (TbGRASP) and the TGN (TbGRIP70).
Here, we have carried out a depletion study on Sec24 paralogs and show that some Golgi components are packaged by only one isoform.
The flagellum attachment zone (FAZ) is a filamentous, cytoplasmic structure in T. brucei that connects the flagellum to the cell body. It has been suggested that, at its
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Figure 1: TbSec24 isoforms in procyclic T. brucei . A) Polyclonal antibodies raised against TbSec24.1 or TbSec24.2 peptides bound, respectively, a 108- or a 101-kDa protein in immunoblots of whole-cell lysates. B) Procyclic trypanosomes were fixed with methanol, stained with anti-TbSec24.1 or anti-TbSec24.2 antibodies and observed by immunofluorescence (left panels) and differential interference contrast (DIC) microscopy (right panels). Nuclear (N) and kinetoplast (K) DNA was labeled with 4’,6-diamidino-2-phenylindole (DAPI). TbSec24.1 and TbSec24.2 typically localize to a structure between the kinetoplast and nucleus. Shown are cells early in the cell cycle with one nucleus and one kinetoplast (1N1K). Bar, 5 μm.
posterior end, the FAZ is in contact with the ERES (39,45). Colabeling with either TbSec24.1 or TbSec24.2 antibodies and the monoclonal FAZ-specific antibody, L3B2, confirmed this. The ERES localized close to the posterior end of the FAZ (Figure 2D).
Knockdown of TbSec24.1 and TbSec24.2 in procyclic T. brucei cells To study the effects of TbSec24.1 and TbSec24.2 depletion, TbSec24.1 and TbSec24.2 RNAi cell lines were generated. Using the inducible, inheritable RNAi system in T. brucei (46), the expression of TbSec24.1 or TbSec24.2 was silenced in procyclic cells by adding doxycycline to induce the expression of the appropriate double-stranded RNA. The specificity of RNAi for both TbSec24 isoforms was tested. TbSec24.1 RNAi was induced for 2 days and samples for immunoblotting were taken every 24 h. While the protein levels of TbSec24.1 decreased, the levels of TbSec24.2 remained the same. The opposite result was obtained in Sec24.2 RNAi cells with TbSec24.1 levels being unaffected by TbSec24.2 RNAi (Figure 3A). The specificity of the RNAi was further corroborated by immunofluorescence microscopy. TbSec24.1 RNAi was induced for 2 days and cells were methanol-fixed and labeled with either TbSec24.1 or TbSec24.2 antibodies. Upon induction of TbSec24.1 RNAi, no TbSec24.1 could be detected. However, the levels and localization of TbSec24.2 were unaltered (Figure 3B). When the reciprocal experiment was performed and TbSec24.2 RNAi was
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induced for 2 days, no change in TbSec24.1 labeling was apparent, whereas TbSec24.2 labeling was undetectable (Figure 3B). The depletion efficiency of TbSec24.1 and TbSec24.2 RNAi was determined by semiquantitative western blotting. This involved serially diluting total cell lysates from control cells to provide a standard for TbSec24 protein levels and fractionating them by SDS–PAGE alongside undiluted samples from induced cells, 24 and 48 h after induction. The Sec24 protein levels in both cell lines decreased, relative to control protein levels, to about 40% after 24 h and to about 15% after 48 h (Figure 3C). Previous experiments have shown that both isoforms of TbSec24 are essential in the BSF of T. brucei (36); therefore, similar experiments were carried out for the PCF of the parasite. The first growth defects could be observed in both RNAi cell lines as early as 2 days post-induction. Cells were arrested in growth around 4 days after addition of doxycycline (Figure 3D). However, significant cell death did not seem to occur. When washed with doxycycline-free medium on day 6 and allowed to grow, duplication of cells was observed after only 1 day, and by day 11 the growth rate was indistinguishable from that of control cells (Figure 3D). At the beginning of the T. brucei cell cycle, cells have one nucleus and one kinetoplast (1N1K). As the cell cycle progresses, the mitochondrial DNA is duplicated and the 1577
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Figure 2: Localization of TbSec24.1 and TbSec24.2 with respect to the ERES, the Golgi and the FAZ. A–C) Procyclic trypanosomes stably expressing Ty-TbSec24.1 or Ty-TbSec24.2 were fixed with methanol and labeled with anti-Ty antibodies. A) The TbSec24 isoforms colocalize with the ERES marker YFP-TbSec13.1 [insets in (A)]. B and C) The TbSec24 isoforms are juxtaposed with the Golgi and the TGN. The Golgi stack was labeled using antibodies to the Golgi matrix protein, GRASP; the TGN was marked using anti-GRIP antibodies. Note that the TbSec24 isoforms are closer to the Golgi stack [insets in (B)] than the TGN [insets in (C)]. D) Methanol-fixed cells were labeled with anti-TbSec24.1 or anti-TbSec24.2 antibodies and the FAZ-specific monoclonal antibody L3B2. Both TbSec24 isoforms localize to the posterior ends of the FAZ (insets). Nuclear and kinetoplast DNA were labeled with DAPI. Bars, 5 μm.
kinetoplast duplicates and segregates giving rise to a cell harboring one nucleus and two kinetoplasts (1N2K). Subsequently, and independent of the kinetoplast, the nucleus divides giving rise to a cell with two nuclei and two kinetoplasts (2N2K). Two daughter (1N1K) cells are generated after cytokinesis. To determine whether TbSec24 RNAi cells accumulate at a specific cell cycle stage, TbSec24.1 or TbSec24.2 RNAi was induced for 3 days and samples were taken every 24 h from control and induced cells. Cells were methanol-fixed, stained with 4’,6-diamidino2-phenylindole (DAPI) and the number of cells in each cell cycle stage was counted. Interestingly, there was no apparent change in the relative number of 1N1K, 1N2K or 2N2K cells in induced RNAi cells when compared with the control cells. Furthermore, there was no accumulation of abnormal cell cycle stages such as 2N1K cells, 1N0K cells, zoids or multinucleated cells (Figure 3E).
Effect of TbSec24.1 and TbSec24.2 depletion on secretion The next step was to determine whether procyclic TbSec24.1 or TbSec24.2 RNAi cells are defective in 1578
anterograde trafficking of proteins, which are transported from the ER, via the Golgi, to the cell surface. Toward this goal the secretion of the soluble secretory marker BiPNAVRG-HA9 was followed. BiPNAVRG-HA9 is an artificial fusion protein comprising 415 amino acids of the BiP-ATPase domain together with the C-terminal hexapeptide QPAVRG and an hemagglutinin (HA)9 epitope tag. In contrast to native TbBiP, BiPNAVRG-HA9 lacks the MDDL retrieval signal and so is not retained in the ER but is efficiently secreted into the medium (47). TbSec24.1 and TbSec24.2 RNAi cells were stably transfected with BiPNAVRG-HA9. TbSec24.1 or TbSec24.2 RNAi was induced for 2 days and cellular proteins were pulse-labeled by incubation with 35 [S]-methionine. BiPNAVRG-HA9 was immunoprecipitated from cell lysates and the medium using HA antibodies after a chase period of up to 6 h. In control TbSec24.1 and TbSec24.2 RNAi cells, the half time (t1/2 ) for cell exit of the BiPNAVRG-HA9 protein was between 60 and 90 min (Figure 4). BiPNAVRG-HA9 was still secreted after induction of TbSec24.1 or TbSec24.2 RNAi but with reduced kinetics. The t1/2 was now
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Figure 3: Depletion of TbSec24.1 and TbSec24.2 using RNAi. A–E) TbSec24.1 and TbSec24.2 RNAi cell lines were induced in the presence of doxycycline (10 μg/mL). A) Total cell lysates (4 × 106 cells/lane) from control (−Dox) and induced cells (+Dox) after 24 and 48 h were separated by SDS–PAGE and immunoblotted using antibodies to the two TbSec24 isoforms, and anti-tubulin was used as the loading control. Representative blots are shown. B) After 48 h of induction, control and induced RNAi cell lines were fixed with methanol and labeled with anti-TbSec24.1 or anti-TbSec24.2 antibodies. Nuclear and kinetoplast DNA were labeled with DAPI. Bars, 5 μm. C) Lysates were prepared from control (−Dox) and induced (+Dox) cells 1 and 2 days post-induction. Those from the control samples were serially diluted (3 × 106 cells equals fraction 1) to permit quantitation of the TbSec24 isoforms in the induced samples after immunoblotting with polyclonal antibodies. Tubulin was used as a loading control. D) TbSec24.1 and TbSec24.2 depletion causes a growth defect. Cells were seeded at a density of 106 cells/mL and samples taken for counting every 24 h. The cultures were reseeded at 106 cells/mL with fresh doxycycline every 48 h. After cell density measurements on day 6, cells treated with doxycycline were washed with the medium, reseeded at 106 cells/mL and cultured in the medium with or without doxycycline. The graph shows the result of three independent experiments. Values are mean ± SD. E) Samples of control and induced RNAi cells were taken every 24 h for 3 days and nuclear (N) and kinetoplast (K) DNA were labeled with DAPI to determine the cell cycle state (normal cell cycle stages, 1N1K, 1N2K, 2N2K; abnormal cell cycle stages, 0N1K, 1N0K). At least 1300 cells were analyzed for each time-point and condition and graphs show the percentage of cells.
between 3 and 4 h (Figure 4). These data suggest that anterograde transport of this specific soluble cargo is partially inhibited in cells depleted of either TbSec24.1 or TbSec24.2.
Effects of TbSec24.1 and TbSec24.2 RNAi on the localization of Golgi proteins To examine the effects of COPII transport on Golgi biogenesis, the localization of various Golgi markers was investigated in TbSec24 RNAi cells. Five different Golgi markers were examined upon induction of TbSec24.1 or TbSec24.2 RNAi. When the localization to the Golgi was analyzed, only the number of cells with visible Golgi foci Traffic 2011; 12: 1575–1591
as determined by immunofluorescence microscopy was scored; the number of Golgi/cell was not assessed. Three distinct phenotypes for the localization of Golgi proteins were revealed upon depletion of TbSec24. The first was partial accumulation of the putative N -acetylD-glucosamine (GlcNAc) transferase, TbGntB, in the ER with a virtually unaltered localization in the Golgi. The steady-state localization of TbGntB at the Golgi was shown previously by immunofluorescence and immuno-EM (38). TbSec24.1 and TbSec24.2 RNAi cells were stably transfected with a TbGntB-YFP construct under the control of the strong constitutive procyclic acidic repetitive protein 1579
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Figure 4: Effect of TbSec24.1 or TbSec24.2 depletion on anterograde transport of secreted cargo. Both TbSec24 RNAi cell lines were stably transfected with a construct encoding a secretory protein (BiPNAVRG-HA9). After 48 h of induction, cells were shifted for 15 min to cysteine/methionine-free medium, pulse-labeled with 35 [S]-methionine for 15 min and samples from cells and the medium were taken at the indicated chase times. Immunoprecipitated BiPNAVRG-HA9 was fractionated by SDS–PAGE, visualized using audioradiography and quantitated using IMAGEJ software. The cell value at zero time was set to 100%. The graph represents three independent experiments. Values are mean ± SD.
(PARP) promoter. Upon induction of RNAi for 2 days, cells were methanol-fixed, stained with DAPI and visualized by fluorescence microscopy. In around 97% of control cells (TbSec24.1 or TbSec24.2 RNAi), TbGntB-YFP localized to the Golgi (Figure 5A,C). However, after induction of TbSec24.1 or TbSec24.2 RNAi, TbGntB-YFP also localized to the nuclear envelope (Figure 5A). This was confirmed by colocalization with the general ER marker, TbBiP (Figure 5B), which localizes to the nuclear envelope as well as the peripheral ER (36,48). Over the 3-day time–course of RNAi induction, the number of cells with ER labeling increased gradually up to 60% in TbSec24.1 or TbSec24.2 RNAi cells (Figure 5C). The number of cells with Golgi labeling only slightly diminished from 100% to around 90% in both RNAi cell lines (Figure 5C). The second phenotype was that TbSec24 depletion had no effect on Golgi localization of TbεCOP or TbGRIP70 (Figure 6A,B). TbεCOP is part of the COPI coatomer and localizes to the rims of Golgi cisternae (44). TbGRIP70 is a TGN golgin that is involved in post-Golgi transport (44). TbSec24.1 and TbSec24.2 RNAi were induced for 3 days and every 24-h samples were taken, methanol-fixed and labeled, respectively, with antibodies against TbεCOP or TbGRIP70. In control and induced TbSec24.1 or TbSec24.2 RNAi cells, around 90–95% of cells displayed TbεCOP and TbGRIP70 localization that resembled Golgi labeling over the entire 3-day time–course (Figure 6A,B). 1580
The third phenotype was a striking, differential effect of TbSec24 isoform depletion on the Golgi localization of TbGRASP and TbGolgin63, which both localize to the Golgi stack and may play a role in Golgi stacking (38,49). Over the induction period of 3 days for both Sec24 isoforms, samples were taken every day, methanol-fixed and labeled with antibodies against TbGRASP or TbGolgin63. TbSec24.1 RNAi led to a dramatic loss of TbGRASP and TbGolgin63 from the Golgi region (Figure 6C,D). Even 1 day after induction of TbSec24.1 RNAi, only 75% of cells showed TbGRASP labeling at the Golgi. This number decreased further to 34 and 25% on day 2 and 3, respectively (Figure 7A). The loss of TbGolgin63 occurred even faster than TbGRASP. Over the 3 days of TbSec24.1 RNAi induction, the number of cells with Golgi-localized TbGolgin63 decreased from 41 to 18% (Figure 7B). However, TbGRASP and TbGolgin63 localization to the Golgi was unaffected after induction of TbSec24.2 RNAi (Figure 6C,D), and in 90–98% of all cells Golgi labeling could still be observed over the 3 days of induction (Figure 7A,B). This loss was not the result of degradation as samples taken from control and induced cells showed unaltered levels of TbGRASP and TbGolgin63 expression (Figure 7C), suggesting that they both require TbSec24.1, but not TbSec24.2, for proper Golgi localization.
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Figure 5: Effect of TbSec24.1 or TbSec24.2 depletion on the localization of the putative Golgi enzyme, TbGntB. A–C) TbSec24.1 and TbSec24.2 RNAi cell lines were stably transfected with a putative Golgi marker (TbGntB-YFP) under the control of the PARP promoter. After 2 days induction (+Dox), cells were fixed with methanol and TbGntB-YFP visualized alone (A) or after labeling with an ER marker (anti-TbBiP antibodies) (B). Note the accumulation of TbGntB-YFP in the nuclear envelope (arrows in A) confirmed by colocalization with TbBiP (arrows in B). Nuclear and kinetoplast DNA were labeled with DAPI. Bars, 5 μm. C) TbSec24.1 and TbSec24.2 RNAi were induced for up to 3 days. Cells were fixed at the indicated times and the percentage of cells showing GntB-YFP at the Golgi or at the ER was quantified. At least 200 cells were analyzed at each time-point. At least three independent experiments were performed. Representative graphs are shown.
The location of neither TbGRASP nor TbGolgin63 could be determined after TbSec24.1 depletion. For TbGRASP, this might reflect the absence of a binding partner in the Golgi (see Discussion). For TbGolgin63, the most obvious location would be the ER, which has a at least fivefold higher surface area than the Golgi (50). Hence, the simplest explanation would be that TbGolgin63 is so diluted in the ER that it falls below the level of detection by fluorescence microscopy. To counter this dilution effect, TbSec24.1 RNAi cells were stably transfected with a construct constitutively expressing YFP-TbGolgin63 from the PARP promoter, yielding about 20-fold overexpression of the YFP-fusion protein (data not shown). In agreement with previously published data (49), the protein localized to the Golgi and most Golgi displayed a ring-like
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shape by fluorescent microscopy, a consequence of the overexpression. After depletion of TbSec24.1 for 2 days, YFP-TbGolgin63 was, as observed before, mostly absent from the Golgi but did accumulate in the ER, as marked with anti-TbBiP antibodies (Figure 6E). These data suggest that TbGolgin63 is packaged in a TbSec24.1-dependent manner into COPII vesicles.
Reversibility of TbSec24.1 depletion on Golgi localization of TbGRASP and TbGolgin63 The growth arrest of TbSec24.1 RNAi cells could be reversed upon doxycycline removal (Figure 3A), so these cells were methanol-fixed and labeled with antibodies against TbGRASP and TbGolgin63, 5 days after removal of doxycycline (day 11), when the growth rate had returned 1581
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Figure 6: Effect of TbSec24 depletion on the location of other Golgi proteins. Following a 2-day induction of TbSec24.1 or TbSec24.2 RNAi (+Dox), cells were fixed with methanol and labeled with (A) anti-TbεCOP, (B) anti-TbGRIP70, (C) anti-TbGRASP or (D) anti-TbGolgin63 antibodies and observed by immunofluorescence microscopy. Note the loss of GRASP and Golgin63 in cells depleted for TbSec24.1, but not TbSec24.2. E) TbSec24.1 RNAi cells were stably transfected with YFP-TbGolgin63 under the control of the PARP promoter. Cells were fixed in 4% paraformaldehyde (PFA) on day 2 after RNAi induction and labeled with anti-TbBiP antibodies to mark the ER. Upon depletion of TbSec24.1, YFP-TbGolgin63 accumulated in the nuclear envelope (arrows). The exposure of YFP-TbGolgin63 in the image of the RNAi-induced sample (+Dox) was slightly increased over the level used in the control cells (−Dox) to better visualize the nuclear envelope. Nuclear and kinetoplast DNA were labeled with DAPI. Bars, 5 μm.
to the same level as in control cells (Figure 3A). Turning off the RNAi machinery led to restoration of TbSec24.1 protein levels nearly identical to those detected in control cells (day 11, data not shown), and TbGRASP as well as TbGolgin63 localized again to the Golgi (Figure 8, +/− Dox). These data confirm that TbSec24.1 is required for proper Golgi localization of TbGRASP and TbGolgin63.
Effects of TbSec24.1 RNAi on different Golgi populations There are two Golgi populations in T. brucei. One is associated with a bilobe structure (51), which is positioned between the kinetoplast and nucleus and can be identified using the pan-centrin monoclonal antibody, 20H5 (52). In the PCF, one lobe is associated with the old Golgi, the other with the assembling new Golgi (51). The second population of Golgi is not associated with this bilobe structure. They are much smaller (about half the size), mostly present at later cell cycle stages (∼0.58 of the cell cycle, G2/M phase, 1N2K/2N2K cells) and there are typically one to two per cell. Their function is unclear though they contain all the markers found in the bilobe-associated Golgi, and, at least late in the cell cycle (G2/M phase, 1N2K/2N2K 1582
cells), comprise stacked cisternae, though the stacking is more disordered (40). To determine whether these two populations respond differently to TbSec24 depletion, cells were depleted of either TbSec24.1 or TbSec24.2 for 2 days and in fixed cells the bilobe and the TGN (TbGRIP70) were labeled. Before depletion (−Dox), TbGRIP70 marked both Golgi populations, either associated with bilobe (Figure 9A,C, arrows) or not (Figure 9A,C, asterisks). After depletion, the population associated with the bilobe remained but the other population had largely disappeared (Figure 9B,D). These qualitative observations were confirmed by quantitation (Figure 9E–J). Before depletion, more than 95% of cells had one or two Golgi associated with the bilobe; 70–75% had one and 20–25% had two, consistent with the role thought to be played by the bilobe in Golgi biogenesis. These percentages changed very little, if at all, after depletion of TbSec24.1 or TbSec24.2 (Figure 9F,I). In marked contrast, the number of Golgi that were not associated with the bilobe changed dramatically. Before
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Figure 7: Quantification of Golgi marker localization after depletion of TbSec24.1 or TbSec24.2. A and B) One, two or three days after RNAi induction, cells were fixed, labeled for the indicated Golgi marker (GRASP or Golgin63) and scored for the percentage of cells showing the marker at punctate foci. At least 200 cells were analyzed at each time-point for each marker. C) Total cell lysates (4 × 106 cells/lane) were also prepared at each time-point and for each marker (with or without induction), fractionated by SDS–PAGE and immunoblotted for each marker as well as tubulin for the loading control. At least four independent experiments were performed. Representative graphs are shown. Depletion of TbSec24.1, but not TbSec24.2, leads to loss of TbGRASP and TbGolgin63 from the Golgi without any change in the protein levels.
depletion, about 40% of cells had one Golgi not associated with the bilobe. Just 1 day after depletion of TbSec24.1, this had dropped to 15% and remained at this level for several days though there did appear to be a slight increase (to about 20%) by the third day (Figure 9G). In TbSec24.2-depleted cells, the number of cells with nonbilobe-associated Golgi was reduced to 22% after the first day and 15% after the second day. Again, a slight increase (to about 20%) was observed by the third day (Figure 9J). Similar results were obtained when ERES was labeled with TbSec24.2 in TbSec24.1-depleted cells or, conversely, with TbSec24.1 in TbSec24.2 RNAi-induced cells (Figure S1). The number of bilobe-associated ERES was virtually unaltered after depletion of either TbSec24 isoform (Figure S1D,G). However, the number of cells with non-bilobe-associated ERES was reduced to 10–20% after induction of TbSec24.1 or TbSec24.2 RNAi (Figure S1E,H).
Role of TbSec24.1 in Golgi morphology Given the suggested role of GRASP proteins and golgins in Golgi architecture (49,53–55), the morphology of the Traffic 2011; 12: 1575–1591
Golgi was observed after depletion of either TbSec24 isoform. Control and induced cells were subjected to high-pressure freezing (HPF) and further processed for EM. The association of the Golgi with the bilobe could not be ascertained at the EM level so the results apply to both Golgi populations. As shown in Figure 10, the control Golgi typically comprised stacks of four to five flattened cisternae with COPI-sized vesicular profiles at the dilated rims. ERESs with associated COPII-sized vesicular profiles were clearly seen on one side of the stack (the cis-side) and the entire region in which the ERES and Golgi were embedded was free of ribosomes (Figure 10A,B). Depletion of TbSec24.1 had a dramatic effect. A minority of the cells (∼30%) displayed relatively normal-looking Golgi (Figure 10C,D). However, the majority of the cells (∼70%) had severely disturbed Golgi morphologies. Stacks were difficult to discern (Figure 10E–H) and there was often a large accumulation of COPI-sized profiles that appeared to be present throughout the Golgi region (Figure 10E–G). The ERES sometimes appeared more 1583
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Figure 8: Reversibility of TbGRASP and TbGolgin63 mislocalization. A and B) TbSec24.1 RNAi cells were induced for 6 days with doxycycline, washed with doxycycline-free medium and then grown for further 6 days in the absence of doxycycline. Samples were taken on day 3 of induced RNAi cells (+Dox) and on day 11 of uninduced cells (−Dox) and from cells after the removal of doxycycline (+/−Dox). Cells were fixed with methanol and the Golgi was visualized using anti-TbGRASP and anti-TbGolgin63 antibodies. Nuclear and kinetoplast DNA were labeled with DAPI. Bar, 5 μm.
distended (Figure 10D,E,H) and there seemed to be fewer COPII-sized vesicular profiles when compared with the controls. In contrast, depletion of TbSec24.2 had only minor effects (Figure 10I–K). Cisternae were stacked with COPI-sized profiles at the rims. In around 20% of the cells, an elevated number of COPI-sized budding profiles could be observed (Figure 10L). ERESs with COPII-sized budding profiles (arrows) were present (Figure 10I–K). These results suggest that TbSec24.1-dependent COPII transport is required for normal Golgi morphology.
Discussion Here, we show that depletion of TbSec24.1, but not TbSec24.2, leads to an almost complete loss of TbGRASP and TbGolgin63 from the Golgi, as assessed by fluorescence microscopy. The loss of both Golgi proteins was observed at the same time as the TbSec24.1 protein levels decreased to 40% of control levels. When the proteins were further reduced, the mislocalization of TbGRASP and TbGolgin63 became more apparent. Restoring the TbSec24.1 levels by turning off the RNAi machinery led to the recovery of their Golgi localization. Together, these data argue that the localization of both Golgi proteins depends directly or indirectly on the presence of TbSec24.1. Interestingly, this loss of Golgi localization was not accompanied by any apparent change in the expression levels 1584
of TbGRASP or TbGolgin63, as measured by immunoblotting. This indicates that both proteins are still expressed but not delivered to the Golgi. In the case of TbGolgin63, a membrane-spanning protein (49), this results in an accumulation of the protein in the ER. The endogenous protein could not be detected, most probably because the ER is fivefold higher in the surface area than the Golgi (50), so dilution might lower the signal below the threshold needed for detection. However, by overexpression of a YFP-tagged copy it proved possible to increase the signal to a level that showed accumulation in the nuclear envelope and the ER, confirmed by colocalization with the ER marker, TbBiP. The question then arises as to the mechanism by which TbGolgin63 is recognized by TbSec24.1 but not TbSec24.2. One possibility reflects the fact that TbGolgin63 has similar properties to SNARE proteins. It is a coiled-coil protein with most of its mass, the N-terminus, projecting into the cytoplasm (49,56). ER-to-Golgi SNAREs are known to bind directly to Sec24 proteins (15); therefore, it is plausible to suggest that TbSec24.1 might have binding sites for TbGolgin63 that are lacking in TbSec24.2. In this model, TbGolgin63 would be captured by direct physical interaction with TbSec24.1 and subsequently delivered by COPII vesicles to the Golgi. TbGRASP, in contrast, is a peripheral membrane protein that interacts with Golgi membranes through an Nterminal myristoyl moiety. This alone is insufficient to target the protein to the Golgi. In mammalian cells, Golgi localization of GRASP55 and GRASP65 not only depends on N-terminal myristoylation but also requires
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an interaction with the golgin proteins Golgin45 or GM130, respectively (57,58). In budding yeast, Grh1p, which is related to GRASP65, requires N-terminal acetylation as well as an interaction with the putative coiled-coil protein Bug1p for Golgi localization (59). Interestingly, Grh1p/Bug1p has been shown to bind to COPII subunits (59,60); therefore, one possibility could be that TbGRASP interacts preferentially with TbSec24.1, but not TbSec24.2, so would not be transported when its binding partner is depleted. Instead, TbGRASP would either remain in the ER or be released into the cytoplasm, in both cases the concentration would be below the threshold detectable by fluorescence microscopy.
affects the Golgi-localized GntB argues that both TbSec24 isoforms can transport TbGntB from the ER to the Golgi.
Mammalian GRASPs are also known to bind to p24 cargo receptors, which bind to Sec24 proteins, so they could act as adaptors delivering GRASPs to the Golgi (30,61,62). They would localize there because mammalian GRASPs only bind to oligomerized p24 (63), which is enriched in the Golgi (55). There are at least six p24 proteins in T. brucei (36) so if that which binds TbGRASP shows a preference for one TbSec24 isoform, this could explain the loss during TbSec24.1 depletion. Although more work will be needed to distinguish between these possibilities, it is worth noting that, to date, only TbSec24.1 displays selectivity in cargo selection (36).
A decrease in the production of COPII vesicles upon depletion of either TbSec24 isoform might explain the apparent loss of those Golgi that are not associated with the bilobe. The TGN marker TbGRIP was associated with the bilobe after depletion, but the population not associated with the bilobe, which was present in control cells, mostly disappeared. This is despite the fact that bilobe and non-bilobe Golgi have all the same components and have associated ERES, again with all the same components. This suggests that the Golgi in the vicinity of the bilobe is more stable than the Golgi not associated with the bilobe. It is tempting to speculate that in PCFs the bilobe itself may play a role in stabilizing this region of the ER and, although we do not as yet know which, if any, components of the ERES interact with bilobe components, such interactions could well stabilize the ERES, so that when levels of TbSec24 decrease, COPII are preferentially recruited to the bilobe ERES, which in turn would stabilize the bilobe Golgi.
Depletion of either TbSec24.1 or TbSec24.2 had no effect on the location of the TGN marker, TbGRIP70, or the COPI subunit, TbεCOP. In yeast and mammalian cells, GRIP domains bind to ADP ribosylation factor (ARF)-related GTPases (64–69), whereas εCOP binds (as part of the coatomer complex) downstream of the small GTPase Arf1 (70–74). If the targets of both GTPases are insensitive to the type of Sec24 needed to exit the ER, this would explain the observations. In contrast, the putative Golgi enzyme TbGntB is a type II spanning protein and so would be assembled in the ER and have to be transported to the Golgi. Although depletion of neither TbSec24 isoform had any dramatic effect on the localization of TbGntB to the Golgi, this depletion led to an enhanced labeling of the ER, in particular of the nuclear envelope. This suggests that there is some diminution of transport, an observation corroborated by the fact that the secretory cargo marker, BiPNAVRG-HA9, is also transported more slowly when either TbSec24 isoform is depleted. The fact that neither depletion significantly
Depletion of TbSec24.1, but not TbSec24.2, had a dramatic effect on the morphology of the Golgi at the level of EM. Golgi stacks appeared more disordered, as did ERES, and there appeared to be fewer COPII-sized vesicular profiles, at least by qualitative observation. In contrast, there appeared to be many more COPI-sized vesicular profiles throughout the Golgi region, which might reflect an inhibition of intra-Golgi COPI vesicle targeting and/or fusion. The nature of this defect will require further work but is clearly dependent on the TbSec24.1 isoform.
One last point concerns the growth arrest seen when either TbSec24 isoform is depleted. Arrest is complete by 4 days yet can be reversed after 6 days with no apparent effect on the growth rate. This suggests that TbSec24.1 and TbSec24.2 are essential for the transport of key cellular components without which the cell is unable to duplicate. However, the absence of these components from their target compartment does not lead to cell death but stasis akin to a G0 -like state, which normally does not exist in T. brucei. Concordantly, no defects in kinetoplast duplication/segregation, mitosis or nuclear duplication/segregation could be detected. This is in contrast to the BSF where depletion of either TbSec24 isoform causes cell death (36). This might be due to
Figure 9: Differential effects of either TbSec24.1 or TbSec24.2 depletion on the localization of TbGRIP70 to Golgi that are associated (or not) with the bilobe. A–D) TbSec24.1 (A and B) or TbSec24.2 RNAi cells (C and D) were induced with Dox for 2 days, fixed with methanol and labeled with antibodies to TbGRIP70. 20H5 monoclonal antibodies (A and B) or monoclonal anti-TbCentrin4 antibodies (C and D) were used to label the bilobe. Nuclear and kinetoplast DNA were labeled with DAPI. DIC images were taken to assess the morphology of the cells. Bars, 5 μm. Control cells (−Dox) are shown in panels (A and C) and cells depleted for TbSec24.1 (+Dox) are shown in panels (B and D). Note that there are two populations of Golgi labeled with TbGRIP70, one associated with the bilobe (arrows) the other not (asterisks) (A and C). This latter population disappears upon induction (+Dox) (B and D). E–J) Quantification of TbGRIP70 localization. Cells were scored for puncta located next to the bilobe or not. At least 500 cells per time-point were analyzed. Note that depletion had no effect on the association of TbGRIP70 Golgi with the bilobe, but there was a marked reduction in the number of TbGRIP70 Golgi that were not associated with the bilobe.
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technical differences (such as different vector systems and different RNAi targeting sequences) or reflect the fact that BSFs commonly exhibit more severe phenotypes than PCFs (75). The reason will remain unclear until these essential components are identified.
Materials and Methods Cell lines All experiments were performed in procyclic T. brucei 29-13 cells, which were derived from a procyclic 427 strain and encode the tetracycline repressor protein and the T7 RNA polymerase (76). Cells were cultured at 27◦ C in SDM-79 medium supplemented with 20% heat-inactivated, tetsystem approved fetal calf serum (Clontech), 6.5 μg/mL haemin, 15 μg/mL G418 and 50 μg/mL hygromycin. To create the stable and inducible TbSec24.1 RNAi cell line, the nt 708-1251 of TbSec24.1 (Tb927.3.1210) were amplified and cloned into the pZJM (77) vector using XbaI. The specific target sequences were chosen using the RNAIT software on the GeneDB website (http://trypanofan.path.cam.ac.uk/software/RNAit.html) (78). The vector was linearized with Not I and transfected into 29-13 cells. The TbSec24.2 RNAi cell line was created in a similar manner using the nt 338-804 of the TbSec24.2 open reading frame (Tb927.3.5420). TbSec24.1 and TbSec24.2 RNAi cells were stably transfected with TbGntB(56)-YFP-pXS2 (38) or BiPNAVRGHA9-pXS5 (47). TbGntB(56)-YFPpXS2 was digested with Nsi I and BiPNAVRGHA9-pXS5 was linearized using XhoI prior to transfection. TbSec24.1 RNAi cells were stably transfected with YFP-TbGolgin63-pXS2 (49), which was digested with MluI. Endogenous replacements of TbSec24.1 or TbSec24.2 with Ty-TbSec24.1 or Ty-TbSec24.2 were carried out as described in the study by Arhin et al. (79). The transgenic BB2-TbSec24.1 or BB2-Sec24.2 cell lines were further stably transfected with Sec13.1-YFP-pXS2 (38). Sec13.1-YFP-pXS2 was linearized by digestion with Nsi I prior to transfection. Stably transfected cells were selected using 5 μg/mL phleomycin (pZJM), 10 μg/mL blasticidin (pXS2, endogenous replacement) or 2 μg/mL puromycin (pXS5). Clonality was achieved by serial dilution.
Antibodies The polyclonal TbSec24.1 antibodies were raised against the peptides WAPQPNQGPGVPFNG (amino acids 30-44) and KPREQHVLYGTEKEK (amino acids 530-544), and the polyclonal TbSec24.2 antibodies were raised against the peptides QHRFEDPHQMQNWGPQG (amino acids 3046) and SGETQAPKGPPQSQGREPR (amino acids 806-824). Each peptide was injected separately into a rabbit. Antibodies against each peptide were separately affinity-purified using SulfoLink Coupling Gel (Pierce) according to the manufacturer’s instructions. All affinity-purified TbSec24 antibodies were used interchangeably in immunofluorescence microscopy and in immunoblotting.
TbGRASP, TbGRIP70, TbεCOP and TbGolgin63 antibodies have been described before (38,44,49). The monoclonal pan-centrin antibody 20H5 was obtained from Jeff Salisbury (Mayo Clinic) (52); the L3B2 monoclonal antibody (80) and the monoclonal anti-BB2 antibody from Keith Gull (University of Oxford); the monoclonal anti-TbCentrin4 antibody from C. L. de Graffenried and the TbBiP antibody from J. Bangs (University of Wisconsin-Madison) (48). We gratefully acknowledge these gifts. The monoclonal HA antibody was purchased from Covance Research Products.
Silencing TbSec24.1 and TbSec24.2 by RNAi TbSec24.1 and TbSec24.2 RNAi cells were seeded at a density of 1 × 106 cells/mL and cultured in the absence or presence of 10 μg/mL doxycycline at 27◦ C. For growth curves, cells were reseeded at 1 × 106 cells/mL with fresh doxycycline as required every 48 h. To test for reversibility, the RNAi cells were washed thrice by induction of doxycycline-free medium on day 6 of the RNAi and were then reseeded at 1 × 106 cells/mL in doxycycline-free supplemented SDM-79. Cells numbers were quantified using a particle counter (Z2 Coulter Counter; Beckman Coulter) and particles of a size between 3 and 10 μm were counted.
Immunofluorescence microscopy and image analysis Cells were washed once with PBS, then centrifuged onto a coverslip before fixation in −20◦ C cold methanol for 5 min (TbGolgin63 labeling) or 9 min (all other cases). Samples were rehydrated with PBS and subsequently blocked with 3% BSA in PBS overnight at 4◦ C or for 1 h at room temperature. Cells spun down onto coverslips were fixed in 4% PFA in PBS for 30 min (YFP-TbGolgin63/TbBiP labeling). Subsequently, coverslips were rinsed twice with PBS and cells were permeabilized for 5 min in 0.25% TritonX-100 in PBS. Next, coverslips were rinsed three times with PBS and samples were immediately blocked with 3% BSA in PBS for 1 h at room temperature. Primary antibodies were diluted in 3% BSA in PBS and coverslips were incubated with primary antibodies for 1 h at room temperature. Coverslips were then washed with PBS, again blocked with 3% BSA in PBS for 20 min and incubated with the appropriate secondary antibodies (Molecular Probes, Invitrogen) for 1 h at room temperature. Subsequent to washes with PBS, cells were treated with 2 μg/mL DAPI for 10 min and mounted using Fluoromount G (Southern Biotec). Epifluorescence images were either obtained using an Axioskop 2 with an Axiocam camera Plan Apochromat 63× numerical aperture (NA) 1.4 oil lens (all from Carl Zeiss, Inc.) (Figures 1 and 2A–C) or an inverted microscope (Axio Observer Z1, Carl Zeiss MicroImaging Inc.) equipped with a PCO 1600 camera (PCO) and using the manufacturer’s drivers in a custom C++ program. Image processing was carried out using IMAGEJ and ADOBE PHOTOSHOP CS3 softwares (Adobe Systems).
Figure 10: TbSec24.1 RNAi cells display an increased number of abnormal-looking Golgi. TbSec24.1 or TbSec24.2 RNAi cells were grown for 2 days in the presence or absence of doxycycline, then subjected to high-pressure freezing, followed by freeze-substitution, fixation and embedding in epoxy resin. Thin sections were imaged and the morphology of Golgi analyzed (see Materials and Methods and text). A and B) Images from TbSec24.1 RNAi and TbSec24.2 RNAi control cells, respectively. Note the stacked cisternal structures (Golgi stack; GS) in a ribosome-free area. Arrows point to likely COPII vesicles budding from the ERES, whereas likely COPI vesicles are present at the cisternal rims (arrowheads) (C–H). Cells depleted of TbSec24.1 show a range of morphologies, from those that look almost normal (∼30%; C and D) to those that lack stacked cisternae and have increased numbers of COPI buds and vesicles (∼70%; E–H). The ERES often appears swollen (E–H). I–L) Cells depleted of TbSec24.2 show mostly normal Golgi with stacked cisternae and clearly visible ERES- and COPII-budding profiles (I–K). Occasionally, an accumulation of likely COPI-budding profiles (arrowheads) can be observed (L). Bar, 250 nm.
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Western blotting For western blotting, 4 × 106 cells were used in each experiment. Cells were lysed in Laemmli buffer and lysates were loaded onto 10% acryl/bis-acrylamide gels and fractionated by SDS–PAGE. Proteins were then transferred to nitrocellulose and probed with the indicated antibodies. As required, immunoblots were stripped using the Restore™ PLUS Western Blot Stripping Buffer (Pierce) according to the manufacturer’s instructions.
Semiquantitative immunoblotting Semiquantitative immunoblots to determine TbSec24.1 and TbSec24.2 depletion efficiency were performed as described (81). Immunoblots were either incubated with anti-TbSec24.1 or anti-TbSec24.2 antibodies. Antitubulin antibody was used as a loading control. Scanned western blots were quantified using IMAGEJ software.
Secretion of BiPNAVRG-HA9 Secretion assays were performed as described for BiPN-HA9 (47). The only significant difference was that BiPNARRG-HA9 was immunoprecipitated using anti-HA antibodies and not anti-TbBiP antibodies. Immunoprecipitates were fractionated by 12% SDS–PAGE. Exposed autoradiography films were scanned and analyzed using IMAGEJ software.
Electron microscopy For HPF, cells were pelleted at 737 × g for 3 min followed by resuspension in 20% BSA in PBS. Enriched cells were transferred onto flat specimen holders and frozen at approximately 2000 bar using an EMPACT highpressure freezer (LEICA Microsystems). Following freezing, the flat specimen holders were transferred to freezesubstitution units (AFS or AFS2; LEICA Microsystems) and placed in Sarstedt tubes, half filled with the frozen acetone containing 2% OsO4 (82). Samples were substituted for 4 days at −90◦ C, followed by a gradual warming schedule up to 0◦ C. After washing in acetone, the samples were infiltrated in mixtures of Agar 100 epoxy resin and acetone at 10◦ C. For final infiltration with pure resin and subsequent heat polymerization, the tablet-like cell samples were transferred to embedding molds. Thin sections of 60–80 nm were cut using an Ultracut S ultramicrotome (LEICA Microsystems), mounted on copper grids, counterstained with uranyl acetate and lead citrate and examined at 80 kV in a JEOL JEM-1210 transmission electron microscope (Jeol Ltd). Images were acquired and analyzed using a digital camera Morada and ANALYSIS FIVE software, ITEM (Soft Imaging System).
Acknowledgments We thank Brooke Morriswood, Christopher de Graffenried and Cynthia He for critical reading of the manuscript. We are further indebted to all past and present members of the Warren lab for discussions and sharing reagents. This work was funded by the University of Vienna and the Medical University of Vienna.
Supporting Information Additional Supporting Information may be found in the online version of this article: Figure S1: Effects of TbSec24.1 or TbSec24.2 depletion on the population of bilobe- or non-bilobe-associated ERES. (A–H) TbSec24.1 or TbSec24.2 depletion was induced for up to 3 days by addition of 10 μg doxycycline/mL. Cells were fixed with methanol, the ERES was labeled with anti-TbSec24.2 antibodies in TbSec24.1 RNAi cells (A, C–E) and with anti-TbSec24.1 antibodies in TbSec24.2 RNAi cells (B, F–H). The bilobe was marked using the monoclonal anti-TbCentrin4 antibody (A–H). Nuclear
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and kinetoplast DNA were labeled with DAPI. DIC images were taken to analyze the morphology of the cells. Bars, 5 μm. In control cells (−Dox), two populations of ERES can be observed. One is associated with the bilobe (arrows) and the other is not (asterisks). The latter mostly vanishes upon depletion of either TbSec24 isoform (+Dox). Representative images from day 2 of induction are shown (A and B). C–H) The localization of the ERES adjacent to the bilobe (D and G) or not (E and H) was assessed. At least 350 cells per time-point were analyzed. Please note that TbSec24.2 localization to bilobe-associated ERES in TbSec24.1-depleted cells and TbSec24.1 in TbSec24.2-depleted cells, respectively, is unaltered whereas it is rapidly lost from the ERES not associated with the bilobe. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.
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