© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd doi:10.1111/tra.12170

Sec16 Determines the Size and Functioning of the Golgi in the Protist Parasite, Trypanosoma brucei Marco Sealey-Cardona1,† , Katy Schmidt1,2,† , Lars Demmel1 , Tatjana Hirschmugl3 , Tanja Gesell4 , Gang Dong5 and Graham Warren1,∗ 1 Max

F. Perutz Laboratories, University of Vienna and Medical University of Vienna, Dr. Bohr-Gasse 9/3, 1030, Vienna, Austria address: Department of Cell Biology and Ultrastructure Research, Center for Anatomy and Cell Biology, Medical University of Vienna, Schwarzspanierstrasse 17, 1090, Vienna, Austria 3 Research Center for Molecular Medicine of the Austrian Academy of Sciences (CeMM), 1090, Vienna, Austria 4 Department of Structural and Computational Biology, Max F. Perutz Laboratories, University of Vienna, Dr. Bohr-Gasse 9, 1030, Vienna, Austria 5 Department of Medical Biochemistry, Medical University of Vienna, Dr. Bohr-Gasse 9/3, 1030, Vienna, Austria 2 Present

∗

Corresponding author: Graham Warren, [email protected]

Abstract The Sec16 homologue in Trypanosoma brucei has been identified and

growth or secretion. Together these data suggest that TbSec16 regulates

characterized. TbSec16 colocalizes with COPII components at the single

the size of the ERES and Golgi and this size is set for optimal growth of

endoplasmic reticulum exit site (ERES), which is next to the single Golgi

the organism.

stack in the insect (procyclic) form of this organism. Depletion of TbSec16

Keywords ER exit site, Golgi, TbSec16, Trypanosoma brucei

reduces the size of the ERES and the Golgi, and slows growth and trans-

Received 3 December 2013, revised and accepted for publication 5

port of a secretory marker to the cell surface; conversely, overexpression

March 2014, uncorrected manuscript published online 10 March 2014,

of TbSec16 increases the size of the ERES and Golgi but has no effect on

published online 4 April 2014

The Golgi is the central sorting hub of eukaryotic cells. Newly synthesized proteins and lipid cargo are transported from the endoplasmic reticulum (ER) to the Golgi in COPII-coated vesicles (1). There the cargo is processed, mostly by modification of bound oligosaccharides, until it reaches the trans-Golgi network (TGN), where it is sorted and transported to the correct destination. In many cells, transport of cargo from the ER to the cis-Golgi network (CGN) occurs at specialized sites, termed ER exit sites (ERES; 2). The ERES and Golgi must, therefore, be functionally, and are also often spatially, connected. This relationship must be coordinated so as to fulfill the cell’s secretory requirements during the cell cycle.

The ERES is a ribosome-free stretch of the ER from which COPII vesicles bud (3,4). COPII assembly is initiated by recruitment and activation of the small GTPase, Sar1, catalyzed by the guanine nucleotide exchange factor Sec12, an ER-resident membrane protein. Active GTP-bound Sar1 associates with the ER membrane and recruits a heterodimeric complex of Sec23 and Sec24 (5). Sec24 is the major cargo selection subunit (6); Sec23 acts as the GTPase activating protein (GAP) for Sar1 increasing the rate of GTP hydrolysis, thereby providing an in-built uncoating mechanism (7). Formation of the prebudding complex, comprising Sar1-GTP, Sec23/24 and cargo, is followed by assembly of the outer COPII coat consisting of heterotetrameric complexes of Sec13 and Sec31 (5).

†

These authors contributed equally to this work.

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In vitro, these five core components are sufficient to induce membrane curvature and generate coated vesicles. In vivo, however, the original screen for secretory pathway components in budding yeast (Saccharomyces cerevisiae) identified Sec16 as an additional, essential component operating at the level of exit from the ER (8). Sec16 is typically a very large, mostly unstructured protein with at least one conserved domain and has been shown to interact with all of the other components needed to assemble COPII vesicles (9–12). These properties gave rise to the idea that Sec16 functions as a scaffold, defining the ERES and concentrating COPII components to facilitate COPII vesicle budding. This idea is supported by other lines of evidence. Sec16 can form oligomeric complexes (13), and is present in sub-stoichiometric amounts compared to all other COPII core proteins (14). It is also depleted in budded COPII vesicles, at least in vitro (15). In vivo, by immuno-EM, Sec16 is found on the ER adjacent to COPII budding profiles, not on them (16). Sec16 has to be present in order for COPII components to be recruited to ERES, though there may be differences between species. In mammals, Sar1 is needed to maintain Sec16 at ER membranes and may also recruit it (17,18); in Drosophila, Sec16 helps to recruit Sar1GTP which in turn recruits the components of the COPII machinery necessary for coat assembly (19). Furthermore, changes in the level of Sec16 affect the levels of COPII components bound to the ERES (14,17) and changes in the cargo load (via physiological stimuli) affect the levels of Sec16 (20). Such a scaffold should help determine the number of COPII vesicles that bud per unit time and so should affect the flux of cargo through the early secretory pathway. In line with this suggestion, depletion of Sec16 results in impaired secretion and fragmentation or loss of ERES in all systems investigated (13,14,17,19). Sec16 overexpression leads to a block or severe reduction of secretory capacity in most species with the exception of budding yeast (Pichia pastoris) where ERES and Golgi morphology appear unchanged and secretion was not explicitly tested (14). Sec16 has also been implicated in the regulation of COPII assembly through negative regulation of the Sar1 GTPase (21,22) and recent work on P. pastoris suggests that this role might be more relevant than a scaffolding activity (23) 614

since the conserved central domain (CCD), which binds COPII components, is not necessary for Sec16 function. In contrast to other organisms, COPII components localize to the ERES even after relocation of Sec16 to ribosomes (23). Since this absence leads to increased turnover of COPII components, the suggestion is that Sec16 acts as a negative regulator of COPII vesicle budding, providing more time for cargo to be captured. Such a kinetic function is not incompatible with a scaffolding function and together they would regulate the flux of cargo through COPII vesicles. The morphological relationship between the ERES and the Golgi depends on the organism. In the simplest case, the ERES is adjacent to the CGN such that budded COPII vesicles can dock immediately with the Golgi. This organization is clear in higher plants, some yeasts and some protist parasites (24–27). In mammals this relation is obscured because the individual Golgi stacks are subsumed into a ribbon-like structure, near to the nucleus, such that vesicular tubular clusters must ferry cargo from peripheral ERES to the central Golgi (28). Nevertheless, even here the linkage between ERES and Golgi can be revealed by the addition of the microtubule-disrupting drug nocodazole (29), or by observing mammalian cells early in telophase, when the individual Golgi stacks reassemble near to ERES (that persist during mitosis) before moving to the juxta-nuclear region (30). It is only in the budding yeast, S. cerevisiae that no clear liaison exists since there appear to be no ERES and Golgi cisternae are dispersed and not stacked (31). This in turn suggests that the morphology of the Golgi depends on the ERES, a suggestion that extends to Golgi biogenesis where a role for the ERES has long been discussed (32,33). As an approach to understanding the relationship between the ERES and Golgi we have been studying Trypanosoma brucei, the causative agent of sleeping sickness in sub-Saharan Africa. Trypanosoma brucei is a flagellated protist with a digenetic life cycle alternating between the bloodstream form in vertebrate hosts including man and the insect (procyclic) form in the Tsetse fly (34). It has a streamlined architecture and the procyclic insect form contains only one ERES and an adjacent Golgi stack at a precise location in the cell between the nucleus and the flagellar pocket (35). COPII subunits have been characterized in T. brucei, including two isoforms each of Sec23 Traffic 2014; 15: 613–629

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and Sec24 (36,37). They form exclusive hetero-dimers (TbSec23.1/TbSec24.2 and TbSec23.2/TbSec24.1), appear to carry different cargoes and together are essential for growth. TbSar1 is also essential (36) and a TbSec13 homologue has been used to study organelle duplication (38). Owing to their simplified cellular architecture trypanosomes have increasingly been used to study Golgi biogenesis (39). During duplication, a new copy of the ERES and Golgi appear next to the old at almost the same time (38). Here, using the procyclic form of T. brucei we have identified and characterized the TbSec16 homologue. TbSec16 localized to ERES at the light- and electron-microscopic level. Knockout or depletion of TbSec16 was found to reduce ERES size and the rate of secretion as well as cell growth, whereas overexpression increased the size of the ERES. Concomitantly the size of the Golgi followed closely the size of ERES. Interestingly, larger ERES and Golgi did not significantly increase the rate of secretion or the growth rate. These data suggest that the size of the Golgi depends on the ERES and that the size and capacity of these two are optimized for the needs of the parasite.

Results Characterization of TbSec16 Sec16 homologs have been identified in all eukaryotes examined so far. It is typically a large protein, >200 kDa, with a CCD (Figure 1A). The nearest organism, in evolutionary terms, for which Sec16 has been characterized, is the fruit fly, Drosophila melanogaster. Hence the protein sequence of the CCD of D. melanogaster (amino acids 1066–1535) was used to probe for a homologue in T. brucei in the TriTryps database and this resulted in a clear candidate (Tb927.3.3850, hereafter TbSec16) with an overall 30% identity and 46% similarity to the D. melanogaster protein. This assignment was corroborated by comparison with sequences of the CCD of other species (Figure S1, Supporting Information). TbSec16 was predicted to be smaller than other Sec16 proteins (147 kDa versus >200 kDa) and lacked the C-terminal conserved domain found in other species. One of the hallmarks of Sec16 is its robust interaction with Sec13. This was shown originally using biochemical and Traffic 2014; 15: 613–629

genetic tools and more recently using structural methods (9,12,16). In budding yeast, the interaction is mediated mainly through the insertion of the 3-stranded β-sheet preceding the Sec16 CCD into the gap of the six-bladed Sec13 β-propeller (12). To provide further evidence that TbSec16 is a true Sec16 homologue with the characteristic element for Sec13 interaction, homology-based structural modeling was carried out on TbSec13 and TbSec16. Homology-based modeling is generally reliable when primary sequences are more than 30% identical (40–42). TbSec13 shares 32.1 identity and 45.1% similarity with its yeast counterpart (Figure S2A), which allows reliable modeling. The homology between TbSec16 and ScSec16, however, is limited, with an identity and similarity of only 15.2 and 29.5%, respectively. Nevertheless, secondary structure prediction by PSIPRED v3.3 (40) strongly suggested that TbSec16 residues 660–703, which are at the N-terminus of the CCD, form three β strands (Figure S2B). The (PS)2 -v2 protein structure prediction server (42) was employed for the modeling using the reported crystal structure of the yeast ScSec16/ScSec13 complex (3MZK.pdb) as the template (12). The results show that, similar to the reported crystal structure (Figure S2C), TbSec13 forms an open six-bladed β-propeller and residues 660–703 of TbSec16 form a 3-stranded β-sheet (Figure S2D). The β-sheet of TbSec16 fits neatly into the gap of the TbSec13 β-propeller in a similar manner to the ScSec16/ScSec13 interaction (Figure S2E). These modeling results demonstrate that structurally TbSec16 could interact with TbSec13 as do their homologues in budding yeast. In order to detect endogenous TbSec16, polyclonal antibodies were raised against combined peptides from the N- and C-termini and affinity-purified on a mixed peptide column. These antibodies recognized a single protein band in western blots of whole cell lysates of ∼180 kDa (Figure 1B). This higher molecular weight was likely due to the predicted elongated structure of this protein reducing mobility during electrophoresis. Immunofluorescence microscopy showed that TbSec16 localized early in the cell cycle to an elongated structure near to the exit site of the flagellum between the nucleus and the kinetoplast (Figure 1C, left panels). Later, as the kinetoplast (containing the compacted mitochondrial DNA) divided, two elongated structures were 615

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Figure 1: Identification of the Sec16 homologue in Trypanosoma brucei . A) Schematic representation of Sec16 homologues from mammalian (Homo sapiens), budding yeast (Saccharomyces cerevisiae and Pichia pastoris), and fruit fly (Drosophila melanogaster ) compared to T. brucei . The black box represents the CCD (∼400 aa) and the gray box the C-terminal conserved domain. Listed on the right are the predicted protein molecular weights in kDa. B) Wild-type cell lysates were fractionated and blotted with affinity-purified polyclonal antibodies to TbSec16 revealing a single band with an estimated molecular weight of about 180 kDa. C) Antibodies to TbSec16 label a punctate structure early in the cell cycle (1N1K), between the nucleus (n) and the kinetoplast (k; mitochondrial DNA), next to the exit site for the flagellum (open arrowheads, see images merged with DIC). Duplication of the kinetoplast (1N2K cells) and nucleus (2N2K cells) is preceded by duplication of the TbSec16-labelled structure. D) The location of the TbSec16 structure was not affected by fusion of the protein either to YFP or Ty1. Insets: 2×-fold magnification of main image. Bars in (C) and (D): 5 μm.

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Figure 2: Characterization of TbSec16 in procyclic cells. A–C) Cells stably expressing YFP_TbSec16 were labeled with polyclonal antibodies against the indicated proteins. Nuclear (n) and kinetoplast (k) DNA was counterstained with DAPI. TbSec16 colocalized with COPII (A; TbSec24.2), was adjacent to the Golgi stack (B; TbGRASP) and further away from the TGN (C; TbGRIP70). Insets: 2×-fold magnification of main image. D) TbSec16 was detected on ultrathin sections of wild-type cells by affinity-purified antibodies followed by gold-conjugated secondary antibodies. Labeling was restricted to the region of the ER adjacent to COPII profiles (open arrowheads in inset). Bars in (A)–(C): 5 μm. Bar in (D): 200 nm. Bar in inset: 100 nm. FP, flagellar pocket, PM, plasma membrane. detected (Figure 1C, middle panels), and this number was maintained during subsequent division of the nucleus (Figure 1C, right panel). This pattern was reminiscent of that seen for the Golgi (38,43). To confirm this observation with co-labeling experiments, the localization of tagged TbSec16 proteins was first verified. Tagged versions of TbSec16, YFP_TbSec16 and Ty1_TbSec16, had to be generated since the antibodies against COPII and Golgi marker proteins were also polyclonal, precluding double-labeling. Monoclonal antibodies are available to both the YFP and the Ty1 tag. As shown in Figure 1D, these tags had no apparent influence on the localization of the TbSec16 protein. Double-labeling experiments were carried out using the ERES marker, TbSec24.2, a Golgi stack marker, TbGRASP, and a TGN marker, TbGRIP70. YFP_TbSec16 almost completely overlapped with labeling for TbSec24.2, a component of the COPII coat (Figure 2A; 37). This was consistent with COPII vesicles budding from the ERES defined by the TbSec16 protein. TbGRASP, predominantly Traffic 2014; 15: 613–629

localizing to the cis- and medial Golgi (38) was detected adjacent to YFP_TbSec16 in agreement with the ERES being next to the Golgi stack in T. brucei (Figure 2B and 27). As expected, TbGRIP70, a marker protein for the TGN was found at an even greater distance from YFP_TbSec16 (Figure 2C; 43). TbSec16 localization was confirmed by immunogold labeling of Tokuyasu sections. Gold particles (10 nm) were only present over that area of the ER adjacent to COPII budding profiles (but not on them) and next to the Golgi stack (Figure 2D). Quantitation showed that labeling for TbSec16 over the ERES was at least 6x-fold higher than over the adjacent vesicles and vesicle buds, and at least 30x-fold higher than the rest of the ER (including the nuclear envelope, NE; Table 1). To investigate which part of TbSec16 is sufficient to localize to the ERES, a series of truncations of TbSec16 with an N-terminal YFP tag was generated (Figure S3A). After verifying their relative expression levels (Figure S3B), their 617

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Table 1: Linear densities of TbSec16 labeling on the ERES, ER, nuclear envelope and vesicle membranes Gold particles (𝛍m) OE OE control RNAi RNAi control cKO cKO control

ERES

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Buds and vesicles

17 13 6.7 10 2.3 11

0.21 0.18 0.041 0.077 0.077 0.024

2.03 1.4 0.38 1.5 0.001 0.88

Gold grains were counted if they were on or within 20 nm of the indicated membrane compartments and expressed as gold particles/μm. The numbers were acquired from more than 20 images for each genotype. cKO, TbSec16 conditional knockout; OE, TbSec16 overexpression; RNAi, TbSec16 RNAi.

intracellular distribution was analyzed in comparison to endogenous TbSec16 (Figure S3A). All constructs that included the CCD plus a small upstream N-terminal region were found to target correctly to the ERES. The necessary domain was narrowed down to amino acids 531–1080 (Figure S3A,C). A comparable domain has been identified in mammalian and Drosophila cells (16,19). Taken together, these data suggest that TbSec16 defines ERES in T. brucei and functions as a true ortholog of Sec16 with an equivalent domain structure, spatial distribution and targeting requirements.

TbSec16 depletion reduces the size of the ERES and Golgi To investigate the effects of TbSec16 depletion, an inducible and inheritable TbSec16 RNAi cell line was generated. Knock down was induced by the addition of doxycycline (10 μg/mL) and depletion of TbSec16 was monitored every 24 h by immunoblotting and by measurement of the growth rate (Figure 3A,B). Using serial dilutions of the zero time point it was possible to show that RNAi lowered the levels of TbSec16 to about 20% of the control after 96 h (Figure 3A). The decrease in TbSec16 was accompanied by a ∼30% reduction in the growth rate over 5 days (Figure 3B) suggesting that TbSec16 has an essential function. Depletion of TbSec16 also led to changes in the ERES and Golgi. The length of the ERES, measured as the largest distance along the three principal axes of 3D rendered objects (see methods), visibly decreased after 4 days of TbSec16 depletion (Figure 3C, top row). Immunogold labeling showed that a significant reduction 618

in the levels of TbSec16 (Figure 3F) correlated with a morphologically shorter ERES (Figure 3D,E). Shortening of the ERES was accompanied by a decrease in the length of TbSec24.2 labeled structures (Figure 3C, middle row) and by an equally reduced TbGRASP staining (Figure 3C, bottom row). The effect of depletion was quantitated by rendering 3D images of ∼200 ERES and Golgi (examples of rendering in Figure S4C,D), determining the maximum length of the respective fluorescence signal after 4 days of TbSec16 depletion. The median length of the TbSec16 structures fell by 19%, from 0.58 to 0.47 μm (Figure 4A). Similarly, the median values for the length of the COPII (TbSec24.2; Figure 4B) and Golgi (TbGRASP; Figure 4C) structures were also reduced by a similar amount, for COPII by 18% (from 0.62 to 0.51 μm), and for Golgi by 15% (from 0.60 to 0.51 μm). The decreased length of all structures was also matched by a reduction in the fluorescence intensity of all labels. Since the number of ERES and Golgi can vary somewhat during the cell cycle, the total fluorescence of all the structures in each cell was measured. After 4 days of induction of RNAi, there was a significant decrease in labeling for TbSec16 (18%), TbSec24.2 (21%) and TbGRASP (20%; Figure 4D,F). Together these data suggest that the level of TbSec16 present in the cell not only influences the size of the ERES but also the Golgi. This effect was even more pronounced in a conditional knockout (cKO) of TbSec16 generated by replacing both endogenous alleles of TbSec16 with sequences of selection markers (blasticidin and puromycin). In order to avoid lethality of TbSec16 deletion and allow detection, a tagged and doxycycline inducible Ty1_TbSec16 construct was stably integrated in the silent rDNA locus. Expression of Ty1_TbSec16 was reduced by more than 95% in the absence of doxycycline (Figure S5A). Growth slowed down dramatically, eventually leading to cell death after 3 days (Figure S5B). Depletion of TbSec16 was confirmed by labeling ERES using anti-TbSec16 antibodies (Figure S5C). After 48 h of doxycycline withdrawal the labeling of Ty1-TbSec16, TbSec24.2 and TbGRASP essentially disappeared (Figure S5D,E). Disappearance of the latter was not the result of degradation since blotting showed, if anything, that there was slightly more TbSec24.1, TbGRASP and TbGRIP after depletion (Figure S6B), Traffic 2014; 15: 613–629

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Figure 3: Depletion of TbSec16 in procyclic cells. Depletion of TbSec16 was induced by addition of 10 μg/mL doxycycline for 4 days. A) Whole cell lysates were fractionated and blotted using polyclonal antibodies to TbSec16 and tubulin as the loading control. Dilutions of the zero time point were used for semi-quantitative assessment of the relative amounts of TbSec16. B) Cells were counted and the number of cell cycles calculated. Note that depletion of TbSec16 reduces the growth rate over 6 days from 12 to 8.4 cycles. C) Induced and control cells were labeled with antibodies against the indicated markers and stained with DAPI. Note that depletion of TbSec16 reduced the size of the ERES and Golgi. Bars: 5 μm. Insets: 2x-fold magnification. D and E) Immunogold labeling (10 nm particles) of TbSec16 on Tokuyasu Sections. D) Control cells; E) TbSec16 RNAi cells after 4 days. F) Quantitation of the labeling intensity presented as a box plot with an overlaid dot plot. Control: n = 29; TbSec16 RNAi: n = 28. Bars in (D) and (E): 500 nm. F, flagella; FP, flagellar pocket. arguing for relocation. This suggests that TbSec16 localizes upstream of COPII and Golgi components and shows that TbSec16 has an essential function.

inducible promoter into the rDNA locus. Addition of TetR led to a maximal overexpression of 4x-fold (Figure 5A). Overexpression resulted in a visible increase in the lengths of the structures labeled by TbSec24.2, TbGRASP and, to

TbSec16 overexpression increases the size of the ERES and the Golgi Overexpression was achieved by integrating an ectopic copy of Ty1_TbSec16 under the tetracycline (TetR) Traffic 2014; 15: 613–629

a lesser extent, TbGRIP70 (Figure 5C). Again, this effect was quantitated (Figure 6). There was a 21% increase in the length of the structures labeled by TbSec16 (from 0.71 to 0.86 μm), a 20% increase in TbSec24.2 (from 0.64 619

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Figure 4: Quantitation of TbSec16 (ERES), TbSec24.2 (COPII) and TbGRASP (Golgi) in TbSec16 RNAi cells. Depletion of TbSec16 was induced by addition of 10 μg/mL doxycycline for 4 days. (A–F) Control and TbSec16 depleted cells were labeled with antibodies to the indicated proteins. Length and fluorescence intensity of ∼200 structures each were measured. Results are presented as box plots with an overlaid dot plot. A–C) Total length and (D–F) total combined fluorescence of all structures in each cell. Differences between the groups are statistically significant at the *p < 0.05 level using Mann–Whitney’s U -test or Wilcoxon’s test. to 0.77 μm) and a 21% increase in TbGRASP (from 0.91 to 1.1 μm; Figure 6A–C). This increase was even more evident when comparing total fluorescence per cell with 37% for TbSec16 labeling, 42% for TbSec24.2 and 44% for TbGRASP (Figure 6D–F). There was variation in the lengths of the structures from cell to cell after Ty1_TbSec16 overexpression for 24 h at 10 ng/mL of TetR. This is illustrated in the gallery of structures shown in Figure S4A,B, labeled for TbSec16 and TbGRASP. Importantly, increases in the length of the TbSec16 structures were tracked by increases in lengths of the TbGRASP structures showing that this relationship 620

holds at the cell as well as the population level. The interdependency of ERES and Golgi size depending of the levels of TbSec16 expression was also seen in 3D in reconstructed and deconvolved movies (see Movies S1–S3) showing that this was not an artifact due to wide field microscopy. The effects of overexpressing TbSec16 were also confirmed using immunogold labeling (Figure 5D,E). Changes in the length were difficult to observe given the random nature of sectioning planes generated. However, there was a clear increase of about 17% in the median number of gold particles over the ERES profiles, similar to Traffic 2014; 15: 613–629

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Figure 5: Overexpression of TbSec16 in procyclic cells. Overexpression of TbSec16 was induced with the indicated concentrations of TetR for 1 day. A) Whole cell lysates were fractionated and blotted using TbSec16 antibodies and tubulin as a loading control to assess the relative amounts of TbSec16. B) Control and overexpressing (OE) cells (10 ng/mL TetR) were counted and the number of cell cycles calculated. Note that a 4x-fold overexpression of TbSec16 had no significant effect on the growth rate over 4 days. C) TbSec16 overexpression was induced using 10 ng/mL of TetR for 1 day. Cells were fixed and labeled (control) or co-labeled (overexpressing) with the indicated antibodies and counterstained with DAPI. Note that overexpression of TbSec16 increased the size of the ERES (TbSec16), Golgi (TbGRASP) and TGN (TbGRIP). Bars 5 μm. Insets: 2x-fold magnification. D and E) Immunogold labeling of TbSec16 on Tokuyasu sections. Cells were fixed, sectioned and labeled with polyclonal antibodies to TbSec16 followed by goat anti-rabbit IgG conjugated to 10 nm gold. D) Control cells; (E) cells overexpressing TbSec16 (10 ng/mL TetR for 1 day). F) Quantitation of gold label presented as a box plot with an overlaid dot plot. Control: n = 30; induced: n = 37. Bars in (D) and (E): 500 nm. F, flagella; FP, flagellar pocket. that observed by fluorescence microscopy. Interestingly, and in contrast to depletion of TbSec16, which inhibited growth, overexpression did not increase the growth rate. Instead, there was no significant effect over a 4-day period (Figure 5B). The effect of TbSec16 levels on secretion Given the different outcomes of depletion and overexpression with respect to the growth rate, the effect on secretion was measured. A soluble secretory marker was Traffic 2014; 15: 613–629

generated comprising the signal sequence for the surface coat protein, procyclin (44) fused to YFP (ssYFP) and stably transfected into the RNAi and overexpression cell lines. TbSec16 knock down and TbSec16 overexpressions were then induced for 4 days and 1 day, respectively. Expression levels of ssYFP were relatively unaffected by depletion or overexpression of TbSec16 (Figure 7A,B). Induced and control cells were pulsed briefly with (35) S-Methionine and chased for the times indicated (Figure 7C,F). Solubilized cells and medium were incubated with antibodies to 621

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Figure 6: Quantitation of TbSec16 (ERES), TbSec24.2 (COPII) and TbGRASP (Golgi) in cells overexpressing TbSec16. Overexpression of TbSec16 was induced for 1 day using 10 ng/mL TetR. Control and overexpressing cells were labeled with antibodies to the indicated proteins. Length and fluorescence intensity of ∼200 structures each were measured. Results are presented as box plots with an overlaid dot plot. A–C) Total length and (D–F) total combined fluorescence of all structures in each cell. Differences between the groups are statistically significant at the *p < 0.05 level using Mann–Whitney’s U -test or Wilcoxon’s test. ssYFP and the isolated immune-complexes fractionated by SDS–PAGE and quantitated using a phosphoimager. Depletion of TbSec16 had a dramatic effect on the secretion of ssYFP, the average half-time almost doubling from 1.5 to nearly 3.0 h (Figure 7C,D). In marked contrast, overexpression of TbSec16 had no significant effect on the half-time for ssYFP secretion of around 1.5 h. This suggests that decreasing the size of the ERES and Golgi decreases secretion but an increase in size has little if any effect.

Discussion Several lines of evidence suggest that the Sec16 homologue in T. brucei has been identified. First, bioinformatic 622

analysis showed that the CCD of TbSec16 was as similar and identical to other Sec16 homologues as to the Drosophila homologue. TbSec16 lacks the C-terminal conserved domain, as does the shorter form of the human Sec16, which is also more similar in size to TbSec16 (116 versus 150 kDa, respectively; 13, 45). The CCD of TbSec16 shows a higher similarity to the CCD of the shorter form of human Sec16 than to the longer form (34% versus 31%). Since the shorter form cannot compensate for the longer form in humans this might indicate that core functions of Sec16 originally resided in the shorter form and additional features of the long version are not needed in T. brucei, which is an early branching eukaryote. Further work will be needed to clarify this point. Second, TbSec16 was found on the ER membranes adjacent to the Golgi at the light Traffic 2014; 15: 613–629

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Figure 7: Effect of depletion and overexpression of TbSec16 on secretion. A and B) TbSec16 RNAi and TbSec16 OE cell lines were stably transfected with a construct encoding a secretory protein, ssYFP and induced to change the levels of TbSec16. C–F) After induction (RNAi: 4 days; OE: 1 day), cells were pulse-chased with 35 [S]-methionine and samples taken at the indicated times. Immuno-precipitated ssYFP was fractionated by SDS–PAGE and quantitated. C and D) TbSec16 depletion. E and F) TbSec16 overexpression. Note that depletion increased the half-time for secretion whereas overexpression had little if any effect. Results from three independent experiments are shown as the mean ± SEM. and electron microscopy levels. Ultrastructurally, TbSec16 labeling was found on ER membranes adjacent to and rarely on COPII budding profiles. It was barely found anywhere else on the ER membrane or in the cell. Third, just as Sec16 is an essential protein, so too is TbSec16. cKO led to cell death in a few days. Last, depletion using RNAi led to a marked increase in the time taken for a secretory marker to leave the cell consistent with an impairment in ER to Golgi transport. Together these data argue strongly that TbSec16 is the Sec16 homologue in T. brucei. Though we have no direct evidence as yet that TbSec16 interacts with other COPII components, as is the case for Sec16 (9–12), homology-based structural modeling shows that the β-sheet of TbSec16 fits neatly into the gap of the TbSec13 β-propeller in a similar manner to that seen for budding yeast Sec16 and Sec13. Furthermore, changes in Traffic 2014; 15: 613–629

the levels of TbSec16 were tracked by at least one COPII component, TbSec24.2. Depletion of TbSec16 using RNAi or cKO led to a decrease in the size of the ERES as well as a decrease in colocalization of TbSec24.2. Conversely, depletion of the isoforms TbSec24.1 and TbSec24.2 had no effect on the localization of TbSec16 (our unpublished data). Overexpression of TbSec16 led to an elongation of the ERES, and an increase in size that was matched by TbSec24.2. Interestingly, this behavior differs from other organisms where overexpression either has no effect on ERES morphology or it leads to fragmentation (9,14,17,19,32). The reasons are presently unclear. The fact that changes in the levels of TbSec16 led to changes in the levels of associated TbSec24.2 but not vice-versa, argues that TbSec16 defines the ERES, acting as a scaffold, as has been suggested by other groups (19,46). 623

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Using conditional overexpression in stable cell lines it was possible to increase the protein levels of TbSec16 by 4x-fold. However, this was only matched by a 37% increase in the amount of TbSec16 in the enlarged ERES. So why is this increase so small relative to the level of overexpression? Either TbSec16, being a peripheral membrane protein, saturates a putative receptor or downstream COPII components limit ERES growth. Both cases would require the amounts of interaction partners to remain unchanged during TbSec16 overexpression. The existence of a Sec16 receptor or membrane adaptor has long been postulated but despite concerted efforts not yet been identified. A genetic screen for changes in ERES morphology in P. pastoris only revealed Sec16 itself (14) and screens for novel interacting proteins in Drosophila (47) did not yield a receptor either. Known proteins, such as Sar1 or Sec12 are unlikely candidates for several reasons. The role of Sar1 might differ between species; it may act upstream of Sec16 in mammals but downstream in Drosophila (17–19); Sec12 concentrates at ERES in P. pastoris (48) but its delocalization does not disrupt ERES formation (49). The other possibility is that downstream COPII components might limit ERES growth. COPII components and Sar1 exist as soluble and membrane-bound pools and we have shown that the total protein amounts of COPII markers do not change after overexpression of TbSec16 (Figure S6A). All that could change, therefore, is the distribution between the soluble and membrane-bound pools and this could explain why there is only a 37% increase. This, however, conflicts with the idea of Sec16 as a scaffold since these downstream components would be limiting the size of the upstream scaffold. This result is more in line with the recent study suggesting that COPII recruits Sec16 to ERES rather than Sec16 organizing ERES and COPII coat formation (23). The precise role of Sec16 and COPII components in regulating the size of the ERES will require further investigation. Changes in the levels of TbSec16 not only affect the size of the ERES but also the Golgi, emphasizing their interdependence. Depletion of TbSec16 led to reduced labeling with the Golgi marker TbGRASP; both the length and size of the Golgi was reduced to the same extent as the ERES. 624

Reduction of both structures was also seen morphologically using electron microscopy. Similarly, overexpression of TbSec16 led to an increase in the length and size of the Golgi, which again tracked the ERES. This was an unexpected result since overexpression of Sec16 in other systems often results in Golgi fragmentation (9,14,17,19). The mechanism underlying this increase Golgi size is unclear but it might reflect increased budding of COPII vesicles from the enlarged ERES as it has previously been suggested that Golgi size might be a direct function of vesicle production rates (50). Golgi biogenesis may also depend on ERES activity (51). The increase in size of the Golgi marked by TbGRASP was accompanied by an increase in labeling for GRIP, the TGN marker, though to a lesser extent. This suggests that the Sec16 influences not only the size of the early Golgi but also the late Golgi as well. The basis of this differential increase in size throughout the Golgi is unknown. It does, however, suggest that T. brucei might prove to be a suitable model to address the issue of intra-Golgi transport, whether by cisternal maturation or stable cisternae, in the future. Changes in the levels of TbSec16 had interesting effects on growth and secretion rates. Depletion led to a slowing of the growth rate and almost a doubling in the half-time for a soluble secretory marker to exit the cell; overexpression had no effect on the growth rate and no significant effect on the half-time for secretion of the marker. These results suggest that the size of the ERES and Golgi are optimal for secretion. Too small and secretion (as well as growth) is affected; too big and there is no effect because there is already sufficient capacity to cope with the secretory load. For a parasite such as T. brucei it may provide an evolutionary advantage to have no spare secretory capacity. After invasion it needs to multiply as fast as possible to help avoid the host response. Doubling the cell includes doubling the plasma membrane, the components of which constitute most of the flux through the secretory pathway in this parasite (52). Perhaps the synthesis of these proteins in the ER somehow signals to TbSec16, which in turn adjusts the size of the ERES and Golgi to cope with this flux. There is evidence in other systems that Sec16 can be regulated by phosphorylation or cargo load (20,53) so this might be a fruitful avenue for future research. Traffic 2014; 15: 613–629

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Materials and Methods Sequence analysis (Position-Specific Iterated BLAST) was performed using National Center for Biotechnology Information server

PSI-BLAST

the

(http://blast.ncbi.nlm.nih.gov). Secondary structure prediction was analyzed with the PREDICTPROTEIN server (http://www.predictprotein.org/). Prediction of protein disorder, domain and globularity was carried out with GLOBPROT 2 server (http://globplot.embl.de/). Alignments were generated using the MAFFT algorithm in JALVIEW (54).

Antibodies

accomplished by double homologous recombination as previously described (57). Briefly, an ectopic copy of the wild-type TBSEC16 N-terminally tagged with Ty1 under TetR control was integrated in the silent ribosomal RNA spacer locus. The following targeting construct was assembled in the pCR4Blunt-TOPO vector: 5′ -500 bp of the TBSEC16 5′ -UTR-blasticidin resistance gene-tubulin intergenic region-500 bp of TBSEC16 3′ -UTR-3′ , and introduced into the T. brucei 29.13 strain. For the second construct, a puromycin resistance cassette was used. Stable transformants were selected by growth in media containing 10 μg/mL blasticidin, 1 μg/mL of puromycin and 7 ng/mL of doxycycline and cloned by limiting dilution. Putative clones were screened for recombination at the correct locus by PCR using genomic DNA, and expression of Ty1_TbSec16 was confirmed by immunoblotting and immunofluores-

Antibodies specific for TbSec16 were raised against two synthetic peptides P1:SPSQGYSPGFVLYTARER (amino acids 269–286) and P2:VNRRMPVRTKYVDTFNSS (amino acids 1370–1387) as haptens

cence analysis. Clones were kept at 5 ng/mL of doxycycline to achieve endogenous expression levels (data not shown).

coupled to keyhole limpet hemocyanin (KLH). Peptide synthesis and immunizations were outsourced (Eurogentec). The peptides were mixed for injections. Antibodies were affinity-purified against the mixed pep-

For ectopic overexpression of TbSec16, Ty1_TbSec16 was cloned into the pGR186 TetR inducible vector (gift from A. Estevez, Granada, Spain).

tides coupled to the SulfoLink Immobilization Kit for peptides (Thermo Scientific) according the manufacturers instructions. Anti-alpha tubulin antibody and anti-BB2 monoclonal antibody were purchased from Roche and Sigma-Aldrich, respectively. Anti-GFP antibody, anti-TbGRASP, anti-TbSec24.1, anti-TbSec24.2 and anti-TbGRIP70. Antibodies have been described before (37,43).

Trypanosome cell lines and cultures All cell lines used for TbSec16 RNAi and overexpression were derivatives of strain 427 (55). Trypanosoma brucei 29.13 cell line was supplied by G. Cross (Rockefeller University). Cells were passaged at 27∘ C in SDM-79 supplemented with TetR-free FBS (10%, Clontech). To study protein localization, the full-length coding sequence of TbSec16 was amplified from genomic DNA by polymerase chain reaction (PCR) and cloned into the pXS2 vector at the 3′ end of the yellow fluorescent protein (YFP; 56). Procyclic 427-1313-514 trypanosomes were transfected with 10 μg of Nsi I linearized plasmid. Stable integration into the tubulin locus of T. brucei was selected using 10 μg/mL of blasticidine. A stable and inducible RNAi cell line was generated by amplifying nt 2069–2569 of the TbSec16 ORF. The target sequence was chosen using the RNAIT software on the GeneDB (http://trypanofan.path.cam.ac.uk/software/RNAit.html).

pGR186 was modified from pGR19 (58) by replacing the EP1 promoter with a T7 promoter under the control of a single TetR operator sequence. Procyclic 427-1313-514 trypanosomes were transfected with 10 μg of Not I linearized plasmid. Stable integration into the ribosomal RNA spacer of T. brucei was selected using 50 μg/mL of hygromycin. Transformants were cloned by limiting dilution. Cells were cultured in SDM-79 with 10% heat-inactivated TetR certified FBS (Clontech) at 27∘ C under the respective drug selections. For TbSec16 overexpression cells were induced using 10 ng/mL of TetR.

Growth curves and immunoblotting For growth curves, cells were reseeded at 1 × 106 cells/mL with fresh doxycycline or TetR as required every 48 h. Cell numbers were quantified using a particle counter (Z2 Coulter Counter; Beckman Coulter). For immunoblotting, cells were washed once in phosphate-buffered saline (PBS) and then lysed directly in SDS–PAGE loading buffer. The equivalent of 4 × 106 cells was loaded per sample and fractionated by SDS–PAGE, transferred to nitrocellulose and detected with the specified antibodies.

Metabolic radiolabeling This method is a modification of (56). About 108 of TbSec16 RNAi

The

p2T7TA blue

vector was linearized with Not I and used to transform

427–1313–514 cells (55). Stable clones were selected by limiting dilution using 50 μg/mL hygromycin. Putative clones were induced with 10 μg/mL doxycycline and analyzed by immunobloting to determine TbSec16 expression silencing, and immunofluorescence to confirm clonal behavior. TbSec16 RNAi cells were seeded at a density of 1 × 106 cells/mL and cultured in the presence or absence of 10 μg/mL

(induced for 4 days) or TbSec16 overexpressing cell lines (induced for 1 day) stably transfected with a construct encoding a secretory protein (ssYFP) were used. ssYFP was constructed by cloning the signal sequence of the first 27 N-terminal amino acids of T. brucei surface coat EP-3 procyclin protein (44) fused to YFP into pHD1034. After resuspension in 1 mL of prewarmed (27∘ C) Met/Cys-free TM-P medium with 10%

doxycycline.

dialyzed fetal bovine serum (Gibco), pulse-labeling was initiated by the addition of 200 μCi/mL [35 S] Met/Cys (EXPRESS35 S, Protein Labeling Mix, Perkin Elmer) for 15 min at 27∘ C. Cells were then chased for the

The cKO cell line of TbSec16 was generated in the background of the procyclic 29.13 strain of T. brucei. Endogenous replacement of both alleles of TBSEC16 with either blasticidin or puromycin cassettes was

indicated times by the addition of 9 mL of prewarmed complete TM-P growth medium. Cells were monitored throughout the labeling period by microscopy.

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Immunoprecipitation

embedded in 12% gelatin. Thin sections of 60 nm were cut with a Leica

About 1 mL of 107 metabolically labeled cells was separated into supernatant and pellet fraction. The pellet fraction containing the cells was

UCT6 ultramicrotome equipped with a Leica EM FCS cryo-setup. All incubations for immunogold labeling were carried out at room temperature. Sections were blocked in 3% BSA, and incubated with anti-TbSec16

solubilized in a buffer with protease inhibitors and detergent. Detergent was added to culture supernatants to the same final concentrations. Cell lysates and supernatants were cleared by centrifugation (15 min, 4∘ C, 17,000 × g.). Normal Rabbit IgG antibody (Invitrogen) and protein A-Sepharose suspension were consecutively added to all samples for preclearance of non-specific protein binding. GFP antibody was added to 1 mL of the precleared supernatants, followed by protein A-Sepharose beads. Precipitated and washed beads were resuspended in 1× SDS loading buffer and fractionated by electrophoresis. Gels were analyzed by phosphorimaging using a Typhoon 8600 scaner (Amersham Biosciences). Quantitative analysis was carried out with IMAGEJ software.

Immunofluorescence microscopy Cells were spun onto glass coverslips (900 × g, 30 seconds) and immersed for 7 min in −20∘ C cold methanol. Cells were rehydrated in PBS and

(1:300 in 3% BSA/0.1% BSA-c) for 1 h. Anti-TbSec16 was detected with goat anti-rabbit IgG conjugated to 10 nm gold (1:20 in 3% BSA; BBI Solutions). Images for ERES measurements were taken at random provided morphologically distinct structures were identifiable. Measurements were taken either from label to label or from one morphologically discernible end of an ERES to the other using the measurement tool in ADOBE PHOTOSHOP.

The data were analyzed using Mood’s Median Test and graphically represented with IBM SPSS STATISTICS version 21.

To calculate the linear densities of TbSec16 label on different membranes, the same set of images was used. ADOBE PHOTOSHOP CS6 was used to measure the ERES length, ER and NE. The vesicle circumference was measured using IMAGEJ. Gold particles were included in the count so long as their distance to the measured membrane was ≤20 nm. Calculations were carried out using MS Excel.

blocked overnight in 3% BSA/PBS. Primary and secondary antibodies were diluted in 3% BSA/PBS. Coverslips were incubated for 1 h at room temperature each. Alexa 488-conjugated chicken and Alexa 594-conjugated goat secondary antibodies (Invitrogen) were used for detection. Coverslips were mounted on glass slides with DAPI Fluoromount G (Southern Biotech) and imaged using a custom-built epifluorescence microscope (Zeiss Observer Z1) equipped with a PCO 1600 camera and a Plan-Apochromat 100×/1.46 oil immersion lens (Zeiss). VISIVIEW version 2.1.1 (Visitron Systems) was used to control the microscope for acquisition. Image processing was carried out using IMAGEJ, ADOBE CS6 PHOTOSHOP

and ILLUSTRATOR (Adobe Systems).

Image analysis Quantification of structure length, surface and fluorescence, were all conducted using Huygens Scientific Image. All images were deconvolved with the HUYGENS PROFESSIONAL software version 4.4.0 (Scientific Volume Imaging) using the measured point spread function (PSF) and the maximum likelihood-estimation algorithm for each channel. The PSF for the green and red dyes were obtained from the image stacks acquired from the green and red 175 nm fluorescent bead samples (Molecular Probes PS-Speck ).

™

For quantification of the fluorescent signals, the Object Analysis tool of the HUYGENS PROFESSIONAL software was used. Fluorescent signals of ERES and Golgi were rendered into a 3D object from which the length, surface and voxels density were obtained. To perform a quantitative analysis, the length, surface and voxel values of all objects were obtained from images that were identically acquired from identically prepared and processed samples. 3D volume renderings were performed using identical threshold and seeding levels for all images. For the statistical analysis and representation the R package was used.

Electron microscopy and immunogold labeling Electron microscopy on ultrathin sections was carried out following the Tokuyasu method (59). Briefly, samples were fixed with 0.2% glutaraldehyde and 2% paraformaldehyde, cryo-protected in 2.3 M sucrose and

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Acknowledgments We thank members of the Warren lab for critical discussions. The MFPL BioOptic Facility for assistance in the image analysis. 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: Alignment of the CCDs of Sec16 homologues. In silico identification of TbSec16. Sequences were retrieved by PSI-BLAST, trimmed to the CCD, aligned using CLUSTALW and visualized by JALVIEW. The columns are colored according to the physicochemical properties of the amino acid using the CLUSTALX color scheme. Figure S2: Homology-based modeling suggests that TbSec16 interacts with TbSec13 in a manner similar to Saccharomyces cerevisiae (ScSec16) and ScSec13. A) Primary sequence alignment of TbSec13 and ScSec13. The sequences were aligned using Muscle WS in JALVIEW. The two proteins share ∼35% sequence identity. Residues are color-coded according to the CLUSTALX Scheme. B) Secondary structure prediction of the N-terminal part of the TbSec16 CCD (residues 660–703). The prediction was carried out using PSIPRED v3.3 (40). C) Known crystal structure of the ScSec16/ScSec13 complex (3MZK.pdb). The ScSec13 beta-propeller is shown in sky blue. For clarity, only the N-terminal beta-sheet of the ScSec16 CCD that interacts with ScSec13 is shown (magenta), with the rest of the CCD (the helical bundles) omitted. D) Homology-based modeling of the TbSec16/TbSec13 complex using the ScSec16/ScSec13 structure as the template. Modeling was carried out on the (PS)2 -v2 protein structure prediction server (42). TbSec13 and the N-terminal beta-sheet of the TbSec16 CCD are shown in cyan and yellow, respectively. E) Two orthogonal views of the in silico model of TbSec16/TbSec13 superimposed onto the crystal structure of the ScSec16/ScSec13 complex. Note that the β-sheet of TbSec16 fits neatly into the gap of

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the TbSec13 β-propeller in a similar manner to the ScSec16/ScSec13 interaction. Figure S3: Mapping the ERES targeting domain of TbSec16. A) Diagram of the domain organization of TbSec16 as predicted by the GLOBPROT program and a schematic representation of the constructs generated. P1 and P2 indicate the position of the synthetic peptides used to raise polyclonal antibodies. The amino acid numbers are shown relative to the full-length TbSec16. Constructs were tagged at the N-terminus with YFP. Localization to ERES is indicated by a plus sign. B) Whole cell lysates of transiently transfected cells were fractionated and blotted with anti-GFP antibody. C) Representative image of the localization of YFP_TbSec16_531-1080 in procyclic cells showing essentially complete overlap with endogenous TbSec16. Insets: 2x-fold magnification of main image. Bar 5 μm. Figure S4: Examples of ERES and Golgi in cells overexpressing TbSec16. Overexpression was induced for 1 day using 10 ng/mL of tetracycline. A) Control and (B) overexpressing cells were co-labeled with antibodies against the Ty1 tag of TbSec16 and TbGRASP. Note that the increase in size and intensity of the ERES is tracked by the Golgi. C) Epifluorescence images. Maximum z-projection of stacks. D) The same stacks after restoration using Huygens deconvolution software. Bars: (A and B) 1 μm; (C and D) 5 μm. Figure S5: Dose dependent depletion of TbSec16 in procyclic cells. TbSec16 conditional KO (cKO) cells were grown with decreasing concentrations of doxycycline to reduce ectopic TbSec16 expression in a graded manner. A) Whole cell lysates were prepared after 2 days and blotted for TbSec16 using tubulin as the loading control. B) Cells were counted over 5 days and the number of cell cycles calculated. C) Cells were labeled with polyclonal antibodies against TbSec16 before (5 ng/mL dox) and after 48 h of TbSec16 depletion (−dox). Open arrowheads indicate dying cells. D and E) Co-labeling of ERES and Golgi using monoclonal antibodies against the Ty1 tag of TbSec16 and polyclonal antibodies against TbSec24.2 and TbGRASP, respectively. Note that after 2 days of TbSec16 depletion the COPII and Golgi signals have essentially disappeared. Bars: 5 μm. Figure S6: Effect of TbSec16 overexpression and depletion on the levels of COPII and Golgi marker proteins. A) TbSec16 OE cells were treated with increasing doses of tetracycline for 24 h. B) TbSec16 cKO cells were induced for the indicated times. In both cases cell lysates were then blotted for the indicated marker proteins using tubulin as the loading control. Note that overexpression or depletion of TbSec16 had little effect on the levels of other COPII or Golgi markers. Movie S1: 3D rendering of a single ERES and Golgi of Trypanosoma brucei procyclic wild-type cells. Procyclic cells stably expressing YFP_TbSec16 were fixed and labeled with antibodies to TbGRASP. Images were processed as described in the Materials and Methods. Movie S2: 3D rendering of a single ERES and Golgi in Trypanosoma brucei procyclic cells after TbSec16 knock down. Cells were grown in the presence of 0.5 ng/mL of doxycycline for 48 h, fixed and the ERES and Golgi double-labeled using monoclonal antibodies to the Ty1 tag on TbSec16 and polyclonal antibodies to TbGRASP. Images were processed as described in the Materials and Methods.

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Movie S3: 3D rendering of a single ERES and Golgi in Trypanosoma brucei procyclic cells overexpressing TbSec16. Overexpression of TbSec16 was induced by adding 10 ng/mL of tetracycline for 24 h. Cells were fixed and double-labeled using monoclonal antibodies to the Ty1 tag on TbSec16 and polyclonal antibodies to TbGRASP. Images were processed as described in the Materials and Methods.

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