Golgins are coiled-coil proteins that have been implicated in the structure and function of the Golgi complex. Here, we identify and characterize a trypanosomal golgin, TbG63, showing that it has a C-terminal membrane anchor and an N-terminus that projects into the cytoplasm. TbG63 in procyclic parasites is localized to the Golgi and interacts with the active, GTP-form of TbRab1A. Overexpression of TbG63 has dramatic effects on Golgi architecture – effects that require the N-terminus – whereas depletion has little, if any, effect on the growth rate. By contrast, in the bloodstream form of the parasite, depletion of TbG63 slows growth, although it has no obvious effect on the transport of a variant surface glycoprotein (VSG) or on Golgi structure. TbG63 might be a useful tool to study the structure and functioning of the Golgi complex.
Cargo proteins, synthesized and assembled in the endoplasmic reticulum (ER), are transported to the Golgi complex for further post-translational modification before sorting to their final destination. The Golgi complex has a unique architecture in many cells, comprising flattened cisternae arranged as stacks, together with associated vesicles. In mammals, these stacks are linked together, forming ribbon-like structures that are often found next to the nucleus and the centrosome (Bonifacino and Glick, 2004; Farquhar and Palade, 1998; Mellman and Warren, 2000).
A number of structural proteins have been implicated in Golgi architecture. The ribbon-like structure requires an intact microtubule and actin network (Beck, 2005; Thyberg and Moskalewski, 1999); the arrangement of individual cisternae into stacks requires the GRASP family of matrix proteins and golgins (Short et al., 2005). Golgins are coiled-coil proteins involved in tethering membranes to each other and to COPI transport vesicles (Gillingham and Munro, 2003). Most golgins bind to small GTPases of the YPT/Rab or the ARF-like (ARL) families, which regulate membrane attachment of their effectors. The physical attachment of golgins to the Golgi membrane is mediated by a variety of different mechanisms. Some golgins bind to Golgi-matrix proteins. For example, GM130 is targeted to the cis-Golgi through C-terminal binding to GRASP65 using a PDZ-like domain (Barr et al., 1998; Barr et al., 1997; Yoshimura et al., 2001). Golgin45 is targeted to the medial Golgi via GRASP55 (Short et al., 2001). Another means of attachment is via other golgins. For example, p115 binds to both giantin in COPI vesicles and GM130 on membranes, thereby helping to target these vesicles to the cis-Golgi (Beard et al., 2005; Linstedt and Hauri, 1993; Malsam et al., 2005).
Giantin is an example of a golgin that is membrane-anchored. Other examples include CASP and golgin84. A common structural feature is an N-terminal, cytoplasmic, coiled-coil domain and a C-terminal transmembrane domain with a Golgi localization signal in the cytoplasmic sequence adjacent to the C-terminal anchor (Short et al., 2005). CASP and golgin84 have a short Golgi-luminal domain whereas giantin has none (Bascom et al., 1999; Gillingham et al., 2002; Satoh et al., 2003). CASP and golgin84 are recently characterized golgins implicated in retrograde transport. CASP in Golgi membranes binds to golgin84 in retrograde COPI vesicles (Diao et al., 2003; Malsam et al., 2005).
In addition to a function in retrograde transport, golgin84 is also involved in the maintenance of Golgi architecture because it has been implicated in cisternal stacking and the generation and maintenance of the Golgi ribbon (Diao et al., 2003; Satoh et al., 2003). Involvement in such diverse activities has made it difficult to assign precise functions to many golgins, particularly in cells that have elaborate Golgi complexes. With this in mind, we began to analyze the Golgi in simpler organisms. One such is the protozoan parasite, Trypanosoma brucei (T. brucei), a unicellular parasitic organism that, in contrast to mammalian cells, possesses only one Golgi stack per cell (Duszenko et al., 1988; Field et al., 2000; He et al., 2004). A number of Golgi proteins have been identified through homology with their mammalian counterparts, but golgins have been particularly refractory because of their extensive coiled-coil regions. The only golgin-like protein in T. brucei identified to date is the GRIP-domain-containing peripheral protein GRIP70 (McConville et al., 2002), which, in mammals, has been implicated in TGN structure and function (Yoshino et al., 2003). To find more golgins and to determine their role in this simplified Golgi complex, we undertook manual inspection of the T. brucei database. Here, we describe the properties of a putative 63 kDa golgin (termed TbG63), attached to Golgi membranes by a C-terminal membrane anchor.
In silico characterization of TbG63
Manual examination of the T. brucei gene database (Trypanosoma brucei GeneDB; http://www.genedb.org/genedb/tryp/index.jsp) revealed a polypeptide with an extended coiled-coil structure and a membrane anchor that appeared to be a promising candidate for a golgin. Given its predicted molecular mass of 63 kDa, the protein was named TbG63 (GeneDB accession no. Tb11.02.4670). Putative orthologues to TbG63 are present in other trypanosomatid protozoa, including Leishmania major and T. cruzi.
Analysis of its amino acid composition revealed that TbG63 is particularly rich in glutamic acid (14.5%) and glutamine (8.8%), which is characteristic for golgins in general. However, no significant sequence similarity to any known golgin could be observed by means of Blast- or hidden-Markov-model-based searches. To support our assumption that TbG63 is a golgin, we proceeded with an exhaustive in silico annotation of TbG63 using the ANNOTATOR software package (IMP Bioinformatics) (Fig. 1A). Almost the entire protein sequence is of low complexity. However, two prominent features stand out: (1) The CAST algorithm (Promponas et al., 2000) identifies a ∼400-aa-long Q-rich domain starting from the N-terminus, spanning seven out of the eight coiled-coil domains detected by the program impCoil (Lupas et al., 1991). (2) The SAPS program (Brendel et al., 1992) identifies a short hydrophobic segment close to the C-terminus, that is likely to constitute a transmembrane domain. Notably, these two features are seen in human golgin84 (Fig. 1B) and are common to all known golgin84 proteins – both in animals and plants (not shown). The golgin84 orthologs known so far, however, share one additional feature not seen in TbG63. Their Q- or E-rich coiled-coil-domains are preceded by a serine-rich stretch of variable length at the N-terminus (Fig. 1B). To identify whether any other of the described golgins resemble the domain structure of TbG63 more closely than golgin84, we performed the ANNOTATOR analysis on all human golgins. Six proteins lack the N-terminal serine-rich domain (GOLGA1, GOLGA6, GOLGA8B, GOLGA8E, GOLGA8G, GOLGB1). Among these, only a single protein, GOLGB1 is in possession of a C-terminal transmembrane domain. GOLGB1, also known as giantin, has been implicated in Golgi structure and function in mammalian cells. It does, however, have a molecular mass that is six times bigger than that of TbG63 (372 kDa vs 63 kDa) so it is unclear whether TbG63 is the giantin ortholog in T. brucei.
One other possibility is that TbG63 is wrongly annotated in the T. brucei genome. Interestingly, a small protein of 26 kDa has been predicted immediately upstream of TbG63 (GeneDB accession no. Tb11.02.4680). This protein contains a serine-rich domain that is missing in TbG63. Recently, evidence emerged that spliceosomal introns do exist in trypanosomes (Mair et al., 2000). Thus, Tb11.02.4680, or part of it, might constitute an exon of TbG63 rather than a gene itself. Alternatively, since trans-splicing is common in trypanosomes, this serine-rich domain might become attached post-transcriptionally to the TbG63-mRNA. However, the electrophoretic mobility of TbG63 is in agreement with its predicted size of 63 KDa, making this hypothesis less likely, although we cannot exclude the possibility that TbG63 is running anomalously, as do many coiled-coil proteins. Further work will be needed to test this hypothesis.
Topology of TbG63
To confirm experimentally that TbG63 is an integral membrane protein, microsomes were prepared from T. brucei YTat cells expressing a N-terminally YFP-tagged version of the protein. These microsomes were washed with buffer, salt, carbonate or both (see Materials and Methods), the pellets and supernatants were analyzed by western blotting and the results were quantified (Fig. 2A). After the salt treatment in HEPES buffer 90-95% of YFP-TbG63 was found in the membrane fraction. When microsomes were washed in carbonate buffer with or without high-salt content, 80% of YFP-TbG63 remained in the membrane fraction. Together, these data show that YFP-TbG63 is an integral and not a peripheral membrane protein (Fujiki et al., 1982). Luminal binding protein (BiP) was chosen as a control because it is a soluble protein within the lumen of the ER. Hence it was resistant to extraction in the presence of high salt but more than 80% was released by carbonate treatment.
As the TMpred program (http://www.ch.embnet.org/software/TMPRED_form.html) assigned very similar scores for the two possible orientations (N-terminal luminal or cytoplasmic) the topology was addressed biochemically. Microsomes from YTat cells expressing an N-terminal YFP-tagged version of the protein were purified and treated with proteinase K (PK) in the presence or absence of Triton X-100 (TX). After stopping proteolysis by PMSF addition, microsomes were analyzed by western blotting and quantified (Fig. 2B). When treated with PK in the absence of TX, more than 80% of the luminal BiP was still intact as detected by western blotting, in contrast to only 2% of the YFP-TbG63. Solubilization of the membranes with detergent resulted in degradation of 80% of BiP, showing that this luminal protein is sensitive to protease once the membrane barrier had been removed. The addition of TX alone (in the absence of PK) had no effect. These observations show that the YFP tag on the N-terminus of TbG63 is accessible to the protease when the membranes are intact, arguing that the N-terminus projects into the cytoplasm.
One concern with these results was that the tag might have altered the topology of the protein and, so, it was important to look at the endogenous protein. This was possible once an antibody had been successfully raised. Microsomes from untransfected parasites were subjected to the same treatments and analyzed by western blotting using anti-TbG63 antibodies (Fig. 2C). Endogenous TbG63 was found to be associated to the membrane fraction after the carbonate wash, and was digested by PK in intact membranes (in contrast to BiP), confirming the results obtained with the recombinant tagged protein. Fluorescence microscopy was carried out to corroborate the biochemical data. TbG63 is present on the Golgi (Fig. 4B) but a YFP-tagged construct lacking the predicted transmembrane domain at the C-terminus (YFP-TbG63ΔTM) showed clear cytoplasmic staining (Fig. 2D). This shows that the C-terminal region of the protein is needed to anchor it to membranes.
Interaction of TbG63 with TbRab1A in vitro
Several mammalian golgins are known to interact with members of the Rab family of small GTPases (Barr and Short, 2003; Short et al., 2005). The interaction of TbG63 with the T. brucei homolog of Rab1A (TbRab1A) (Dhir et al., 2004) was therefore tested. Recombinant GST-tagged TbRab1A, immobilized on glutathione-Sepharose beads, was used to assay binding to TbG63. Point mutants of TbRab1A that stabilize the protein in the active, GTP-bound [GST-Rab1A (QL)] or inactive, GDP-bound [GST-Rab1A (SN)] conformations were used, and pre-loaded with either GTPγS or GDP, respectively, together with a recombinant soluble His-tagged TbG63 mutant lacking the transmembrane domain (TbG63ΔTM). As shown in Fig. 3, ∼10% of TbG63 bound preferentially to the GTP form of the mutant TbRab1A, which was more than fivefold greater than the amount bound to the GDP form. For the wild-type TbRab1A, the form loaded with GTPγS bound nearly twice as much TbG63 than the form loaded with GDP. No TbG63 was detectable in the control IP with GST-beads alone. These data show that, in common with other golgins, TbG63 binds a Golgi Rab GTPase.
TbG63 is a Golgi resident protein
Since the properties of TbG63 are consistent with it being a golgin, its localization in T. brucei was determined. An enriched Golgi fraction was purified from parasites stably expressing the Golgi marker, GntB-YFP, a putative GlcNAc transferase (Ho et al., 2006; West et al., 2004). Cells were homogenized using a French press and subjected to differential centrifugation to isolate a microsomal fraction (P2; Fig. 4Ai). This was further fractionated using isopycnic centrifugation with a sucrose-step gradient (Fig. 4Aii). The Golgi fraction equilibrated at 28% sucrose, and was tenfold enriched for GntB-YFP compared with the homogenate. Another Golgi marker, ϵ-COP (Ho et al., 2006; Maier et al., 2001), also cofractionated with GntB-YFP. TbG63 cofractionated with these two Golgi markers but there was also a `tail' of higher density, suggesting the presence of TbG63 in ER membranes. The presence of BiP at similar densities is consistent with this interpretation.
To confirm these results morphologically, immunofluorescence studies were performed using antibodies raised against the cytoplasmic domain of recombinant TbG63. Cells stably expressing YFP-GntB were methanol-fixed and labeled with anti-TbG63 antibody (Fig. 4B, upper panels). The colocalization of the YFP-GntB with endogenous TbG63 suggests that this protein is indeed localized to the Golgi. To determine which part of the Golgi contains TbG63, cells stably expressing the trans-Golgi network (TGN) marker YFP-GRIP (Ho et al., 2006; McConville et al., 2002; Munro, 2005) were methanol-fixed and labeled with anti-TbG63 antibody. YFP-GRIP was positioned adjacent to TbG63 (Fig. 4B, lower panels) showing that TbG63 is present in the Golgi stack and not the TGN.
Overexpression of TbG63 affects Golgi morphology
Since mammalian golgins are known to have a role in Golgi architecture, we tested whether TbG63 has a similar role by overexpressing YFP-tagged full-length (YFP-TbG63) and truncated (YFP-TbG63ΔN) proteins in procyclic cells. Both proteins localized to the Golgi complex, since they colocalized with the Golgi marker GRASP (Fig. 5A). Expression of the tagged, full-length protein had a striking effect upon Golgi structure, converting staining of the Golgi complex from punctate (Fig. 5A, first row of panels) to doughnut-shaped (ring-shape) (Fig. 5A, second row of panels). One possible explanation is that this shape was caused by the large size of the GFP tag; therefore, a smaller tag, the ten-amino-acid-long virus-derived BB2 sequence, was used (Bastin et al., 1996). However, the same morphological change was observed (Fig. 5A, third row of panels) showing that the effect is the result of TbG63 overexpression. The doughnut-shaped phenotype was independent of the expression levels of the protein. The effect was further confined to the N-terminal part of TbG63 because a construct lacking this part still colocalized with the GRASP Golgi marker but did not affect the punctate appearance of the Golgi (Fig. 5B). Furthermore, the TGN, in addition to the Golgi stack, is affected as shown using the TGN marker GRIP (Fig. 5A bottom row). The morphological changes were not the result of the fixation procedure because live cell imaging of cells co-transfected with GRASP-mRFP and YFP-TbG63 yielded similar doughnut-shaped structures (data not shown).
Cells expressing YFP-TbG63 were next examined at the ultrastructural level. After transfection and selection, cells were polyclonal and ∼50% stably expressed YFP-TbG63 – as estimated by FACS analysis (data not shown). When these polyclonal cells were fixed, labeled with antibodies against GFP and processed for immuno-electron microscopy, about half of them did not show any gold particles localized over the Golgi complex (non-expressing cells). These non-expressing cells showed the normal morphology of the Golgi, comprising four to five stacked cisternae flanked by an ER exit site (ERES) on one side and the TGN on the other (Fig. 5C, Control). The other half of the cell population showed levels of gold labeling that appeared to correlate with changes to the morphology. At the lowest gold-labeling levels the Golgi complex assumed an arciform morphology (Fig. 5C, Arciform; about 17% of the population). At higher gold-labeling levels there were fewer cisternae and more associated vesicles (Fig. 5C, Vesiculated Cisternae; about 33% of the population). At highest gold-labeling levels the Golgi was converted into tubulo-vesicular clusters (Fig. 5C, Tubulo-vesicular; about 50% the population).
Depletion of TbG63
Depletion experiments were performed using the inheritable RNA interference (RNAi) system in T. brucei to determine whether TbG63 affects the viability or growth rate of the cells (Wirtz et al., 1999). Expression of double-stranded RNA (dsRNA) targeting TbG63, induced by the presence of doxycycline, lowered the endogenous levels of TbG63 about 40% as measured by quantitative western blotting. There was no apparent change in the growth rate of the cells. Immunofluorescence analysis confirmed the depletion of TbG63 but there were no significant changes in the amounts of other Golgi markers or in the shape of this organelle; the example shows ϵ-COP (Fig. 6), and similar staining patterns were obtained for GRASP and GRIP (data not shown). Similar experiments were carried out on the bloodstream form of the parasite, which contains slighter lower levels of TbG63 (data not shown). Importantly, RNAi lowered these levels to about 20% of control values (Fig. 7B) and this led to a decrease in the growth rate of ∼50% (Fig. 7A). Further studies were then carried out to determine whether this slower growth rate could be attributed to changes in Golgi structure and function.
Functional experiments were carried out by measuring the transport of a variant surface glycoprotein (VSG,) which accounts for more than 90% of the proteins at the cell surface. A pulse of [35S]-labeled methionine, followed by a chase, was used to follow the transport of newly-synthesized VSG from the ER to the cell surface via the Golgi. Appearance at the cell surface was measured by release of VSG after hypotonic lysis, which activates a surface phospholipase severing the GPI anchor that tethers the VSG. As shown in Fig. 8, the time course for release of [35S]-labeled VSG was the same, even though depletion of TbG63 exceeded 80%. Morphological experiments also showed no effect on the structure of the Golgi (not shown) following TbG63 depletion. Together, these data argue that the decrease in growth rate cannot be attributed to changes in the Golgi structure or the VSG transport.
By using the T. brucei database, we have been able to identify and characterize the first membrane-anchored golgin in this organism. TbG63 has a predicted Q-rich domain starting from the N-terminus up to the seventh coiled-coil domain and a short hydrophobic segment, a membrane anchor at the C-terminus. What remains unclear is the relationship of TbG63 to golgins that have been characterized in mammalian cells. On the one hand, TbG63 resembles golgin84, even though it lacks the serine-rich N-terminal domain found in all golgin84 proteins studied to date. On the other hand, it is most similar in domain structure to giantin; but the latter has a six times higher molecular mass. Further work is clearly needed, but these in silico analyses argue strongly that TbG63 is a member of the golgin family.
Biochemical experiments confirmed that TbG63 is an integral membrane protein, because it is resistant to extraction with carbonate and salt but not detergent. Deletion experiments showed that the C-terminus is essential for membrane attachment. The topology was investigated using proteases, which had no effect on a luminal membrane marker, the ER protein, BiP. They did, however, degrade endogenous TbG63, and an N-terminally GFP-tagged construct, showing that the N-terminus projects into the cytoplasm. Many golgins are also known to bind to small GTPases of the YPT/Rab and ARL families (Barr and Short, 2003). Pull-down experiments showed that TbG63 bound to the active (GTP) form of TbRab1A. Fractionation experiments showed that TbG63 cofractionated with Golgi markers (GRASP and ϵ-COP), although a small fraction was present in denser membranes, perhaps that of the ER. This would either be consistent with the idea of newly-synthesized TbG63 being on its way to the Golgi, or with the possibility of a recycling pool.
The cellular localization of TbG63 was further confirmed by fluorescence microscopy, showing clear colocalization of TbG63 and the Golgi-stack-marker GRASP. TbG63 did not colocalize with the TGN-marker GRIP but was found adjacent to it. A similar arrangement has been observed previously for GRASP and GRIP (He et al., 2004). Immuno-EM studies were carried out to confirm the localization at the EM level. Antibodies against the endogenous protein did not result in detectable labeling, so, lines stably expressing YFP-tagged TbG63 were used. Unexpectedly, expression of YFP-TbG63 had a dramatic effect on Golgi architecture, converting stacks into a range of vesiculated structures. These structures appear doughnut-shaped when viewed by fluorescence microscopy. They were not the consequence of the YFP tag because the BB2 tag had the same effect, but they did depend on the N-terminal cytoplasmic domain (the first 250 amino acids) because expression of a construct lacking this domain had no effect on Golgi morphology. Interestingly, this domain resembles the BAR domain that binds to curved membranes and, in some instances, can induce curvature (Itoh and De Camili, 2006). This raises the interesting possibility that the doughnut-shaped structures seen upon overexpression are an exaggeration of the normal function of TbG63. Perhaps TbGG63 is needed to help form the COPI vesicles that mediate intra-Golgi transport, a possibility that will require further experimentation.
The polyclonal nature of the overexpressing cells precluded quantitative analysis of the effect of TbG63 on Golgi function. These overexpression experiments were, therefore, complemented by depletion experiments; TbG63 levels were lowered using RNAi. No differences in growth rate were observed and there were no obvious changes in Golgi morphology in the procyclic form. This might, however, reflect the fact the levels of TbG63 could not be lowered below 60%. Similar experiments were then carried out using the bloodstream form of the parasite and, in this instance, it was possible to lower the endogenous levels below 20%. Bloodstream parasites depleted of TbG63 grew only half as fast as the wild-type control parasites, but there was no slow-down in the transport of a VSG to the cell surface and no obvious changes in Golgi morphology, as investigated by EM analysis. One possible explanation is that TbG63 is not involved in the transport of VSG but affects the transport of other cargos. It will therefore be important to extend these studies to other proteins destined for the cell surface or other cellular compartments. In addition, it will be important to knock out the TbG63 gene in both the procyclic and bloodstream forms of the parasite to determine whether it is an essential or a non-essential golgin.
In summary, we have identified and characterized the first membrane-anchored golgin in T. brucei, and show that (when overexpressed) it has a role in Golgi architecture and in cell growth – at least in the bloodstream form. Similar and related approaches should uncover more golgins, leading eventually to a more precise understanding of their role.
Materials and Methods
Cell culture and transgenic expression
Procyclic T. brucei rhodesiense YTat1.1 were used for recombinant reporter expression. These cells were grown in Cunningham medium containing 15% heat-inactivated fetal calf serum (Gemini Bio-Products) at 28°C. The procyclic 29.13 cell line (T. brucei brucei) was used for the RNAi studies. These cells were grown in Cunningham medium with 15% heat-inactivated Tet-system approved fetal calf serum (BD Biosciences) in the presence of 15 μg/mlG418 and 50 μg/ml hygromycin at 28°C. Bloodstream-form parasites (BSF-SM) were grown in HMI-9 medium at 37°C supplemented with 10% Tet– serum and 3 μg/ml G418 to maintain the RNAi machinery. Cells were grown and transfected as described previously (Fantoni et al., 1994).
The T. brucei TbG63 (Tb11.02.4670) sequence was obtained through GeneDB database (http://www.genedb.org/genedb/Search?name=Tb11.02.4670&organism=tryp). The full-length coding sequence (1-544 aa) or shorter constructs (1-500 aa or 250-544 aa) of TbG63 were fused to the C-terminus of GFP by cloning the full-length coding sequence into the pXSGFPM3FUS vector (Bangs et al., 1996; Marchetti et al., 2000). The 1-500 aa TbG63 construct was also fused to the C-terminus of GST by using the pGEx-6P-1 vector and was His-tagged using the pEt30a+ vector.
A 575 bp fragment corresponding to nucleotides 864-1437 of TbG63 was cloned into the pZJM vector to deplete TbG63 in T. brucei cells (Wirtz et al., 1999). Doxycycline (10 μg/ml) was added to induce expression of dsRNA.
Exponentially growing cells were diluted to 1-3×105 cells/ml with fresh medium and cells were counted every 24 hours. The cell density was maintained between 1×105 and 1.5×106 cells/ml. The doubling number was calculated as described (He et al., 2004).
Polyclonal antibodies against TbG63 were raised in rabbits against aa 1-500 fused to GST. The His-tagged fusion TbG63 aa 1-500 was expressed, purified according to manufacturer's instructions (Amersham Biosciences and Quiagen) and used for affinity purification of the TbG63-specific antibodies. Anti-BiP antibody was a kind gift from J. D. Bangs (The University of Wisconsin, WI).
Purification of microsomes and enriched Golgi fraction
The biochemical fractionation was adapted from (Grab et al., 1987). T. brucei cells (0.5-1×1010 cells) were harvested by centrifugation and washed twice in 10 ml H buffer (0.25 M sucrose, 50 mM HEPES-KOH pH 7.4, 25 mM KCl, 5 mM MgSO4) supplemented with one tablet inhibitor cocktail and 42 μg/ml PMSF and resuspended in 20 ml H buffer. Cells were homogenized using a French press, at ∼2500 psi and a 6-minute passage time. The homogenate was then centrifuged at 15000 g for 20 minutes to generate a pellet (P1) and a supernatant (S1) that was further centrifuged at 125,000 g for 90 minutes (overlaid on a two-step gradient: 500 μl 0.3 M sucrose, 50 μl 2 M sucrose) to generate a final microsomal pellet (P2) and supernatant (S2). The microsomes (P2) were resuspended in H buffer by five up-down strokes in a loose-fitting dounce homogenizer. P2 was further fractionated by isopycnic sucrose-gradient centrifugation by layering it on a step gradient comprising 200 μl 25%, 1 ml 32%, 1 ml 39%, 1 ml 42%, 1 ml 50% (w/w) sucrose in H buffer. Membranes were centrifuged to equilibrium at 150,000 g for 16-18 h in a SW55 rotor. 0.5 ml fractions were collected from the top of the gradient, snap-frozen and stored at –80°C until use.
Biochemical determination of TbG63 topology
Microsomes from cells stably expressing YFP-TbG63 or from untransfected cells were prepared as described above. Microsomes (50 μl: 2×108 cell equivalent per assay) were diluted 10-fold with H buffer or 100 mM Na2CO3. After rotation for 80 minutes at 4°C, KCl was added to a final concentration of 1M to half the samples in each set and water to the rest. After 40 minutes rotation at 4°C, they were centrifuged for 40 minutes at 100,000 g. The supernatants were siphoned off and stored and the pellets were resuspended in H buffer to the same final volume as the supernatants. Samples were snap-frozen and stored at –20°C. The presence of YFP-TbG63 or endogenous TbG63 in the membrane fraction after the washes was analyzed by western blotting using anti-GFP antibodies or anti-TbG63 antibodies. As a control, western blotting against the luminal ER protein BiP was also performed.
To determine the orientation of TbG63 in the membrane, 25 μl of microsomes (8.5×107 cell equivalents per assay) was incubated with or without proteinase K (50 μg/ml) in the presence or absence of Triton X-100 (0.5%) in a final volume of 40 μl (adjusted using H buffer). After 35 minutes on ice, 15 μl of 4 mM PMSF (fresh) was added to stop proteolysis. Samples were snap-frozen and stored at –20°C. The presence of YFP-TbG63 or endogenous TbG63 was analyzed by western blotting using anti-GFP or anti-TbG63 antibodies. As a control, western blotting against the luminal ER protein BiP was also performed.
Soluble, recombinant His-TbG63 was incubated with the `GTP' (Q-Rab-GST)-locked and `GDP' (S-Rab-GST)-locked mutants of TbRab1A, GST alone or wild type TbRab1A (wt-Rab-GST). The Rab proteins were preloaded with either GTPγS or GDP. Bound proteins were eluted with GDP/EDTA as described in (Beard et al., 2005).
VSG exocytosis assay
The assay was performed as previously described (Allen et al., 2003).
T. brucei cells attached to coverslips were fixed in and permeabilized with –20°C methanol for 8 minutes, blocked with 3% BSA-PBS before antibody staining. Alternatively, cells were fixed in 4% PFA-PBS for 20 minutes washed twice for 5 minutes in PBS and permeabilized using 0.25% Triton X-100 for 20 minutes at room temperature prior to blocking with 3% BSA-PBS. Anti-Tb-GRASP and Anti-GRIP were used to stain the Golgi complex and the TGN, respectively. DAPI was used at 2 μg/ml to visualize the DNA in the nucleus and the kinetoplasts. Fixed cells were observed using an upright microscope (model Axioplan2; Carl Zeiss MicroImaging, Inc.) equipped with a CCD camera (model Orca-II; Hamamatsu) and a Plan-Apochromat 100× 1.4-NA DIC objective. Images were acquired and processed using Openlab software (Improvision).
Log-phase T. brucei procyclic cells (YTat1.1), stably expressing YFP-TbG63, and procyclic-form cells in which RNAi had been induced for 72 hours were harvested by centrifugation and fixed with 2% paraformaldehyde, 0.2% glutaraldehyde and 0.26 M sucrose in 100 mM HEPES, pH 7.2 and processed further for cryosectioning. 70-nm-thick cryosections were cut using a Leica UCT cryo-ultramicrotome. The sections were labeled with a polyclonal anti-GFP antibody (Seedorf et al., 1999) followed by 10-nm proteinA-gold (Department of Cell Biology, University of Utrecht, the Netherlands). After contrasting with uranyl acetate, the sections were examined using a Tecnai 12 electron microscope (FEI).
SDS-PAGE and western blotting
Protein gel electrophoresis was performed using gradient 4-15% SDS-PAGE gels. For western blotting, proteins were transferred onto nitrocellulose membranes (Millipore), incubated with antibodies [anti-TbG63, anti-GFP, anti-PAR, anti-BiP, anti-tubulin (dilution 1:20,000), anti ϵ-COP] and antigens were detected using the ECL system (Amersham Biosciences). ImageJ 1.38× software was used to quantitate the intensity of the bands and calculate the % of depletion in RNAi cells.
- Accepted February 7, 2008.
- © The Company of Biologists Limited 2008