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First published online 14 April 2008
doi: 10.1242/jcs.014324

Journal of Cell Science 121, 1538-1546 (2008)
Published by The Company of Biologists 2008
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TbG63, a golgin involved in Golgi architecture in Trypanosoma brucei

Irene Barinaga-Rementeria Ramirez1, Christopher L. de Graffenried2, Ingo Ebersberger3, Jordan Yelinek2, Cynthia Y. He4, Albert Price5 and Graham Warren6,*

1 Faculty of Life Sciences, University of Manchester, The Michael Smith Building, Oxford Road, Manchester, UK
2 Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA
3 Center for Integrative Bioinformatics Vienna, Max F. Perutz Laboratories, Dr Bohr Gasse 9, A-1030 Vienna, Austria
4 Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543
5 VaxInnate, New Haven, CT, USA
6 Max F. Perutz Laboratories, University of Vienna, Medical University of Vienna, Dr Bohr Gasse 9, Vienna, 1030, Austria


Figure 1
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  		Fig. 1. Structural and compositional characteristics of (A) TbG63 (Tb11.02.46.70) and (B) human golgin84, as predicted by the ANNOTATOR software package (http://imp6.annotator.org/). {alpha}-helical and β-sheet domains in the annotation line `Secondary structure features' were abbreviated with {alpha} and β, respectively. A and C in the `Small sequence motifs' annotation line represent predicted Asn-glycosylation sites and phosphorylated cAMP sites, respectively.

 

Figure 2
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  		Fig. 2. TbG63 is an integral membrane protein with the N-terminus exposed to the cytoplasm. (A) Microsomes from cells stably expressing YFP-TbG63 were washed in HEPES or carbonate buffer in the presence or absence of 1 M KCl. The supernatant (S) and pellet (P) fractions obtained after high-speed centrifugation were analyzed by SDS-PAGE followed by immuno-blotting for YFP and the ER luminal marker, BiP. (B) The same microsomes in (A) were also treated with proteinase K (PK) in the presence or absence of Triton X-100 (TX) and blots probed for YFP and BiP. For both (A) and (B) a representative blot is shown above and quantitation below presented as the mean ± s.d. (n=3). (C) Microsomes from YTat cells were treated as described in A and B, and blots probed for endogenous TbG63 and BiP. (D) Cells transiently transfected with YFP-TbG63 lacking the C-terminus, which includes the predicted transmembrane domain, were fixed and DNA stained using DAPI.

 

Figure 3
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  		Fig. 3. TbG63 is a binding partner for TbRab1A. Soluble, recombinant His-TbG63 (His-TbG{Delta}TM) was incubated with the GTP-locked (Q-Rab-GST) and GDP-locked (S-Rab-GST) mutants of TbRab1A (lanes 1 and 2), GST alone (lane 3), or wild type TbRab1A (wt-Rab-GST) (lanes 4 and 5). The Rab proteins were preloaded with either GTP{gamma}S (lanes 1, 4) or GDP (lanes 2, 5). Bound proteins were eluted with GDP-EDTA and fractionated by SDS-PAGE followed by immuno-blotting for TbG63 (upper panel) or GST (lower panel).

 

Figure 4
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  		Fig. 4. TbG63 localizes to the Golgi complex. (A) Biochemical fractionation. (i) Cells (1x1010) stably expressing the Golgi marker YFP-GntB were homogenized (H), centrifuged at medium speed to generate a pellet (P1) and a supernatant (S1) that was then centrifuged at higher speed to generate a final microsomal pellet (P2) and supernatant (S2). (ii) The microsomal pellet (P2) was further fractionated by isopycnic sucrose gradient centrifugation. For both i and ii equal fractions were analyzed by blotting for the indicated markers and the results quantitated and presented as the mean ± s.d. (n=4). Blots were probed for: Golgi (YFP-GntB), COPI coats ({epsilon}-COP), flagella (PAR), ER (BiP), tubulin (Tub) and TbG63. (B) Fluorescence microscopy. Cells stably expressing YFP-GntB (top row) or YFP-GRIP (bottom row) were fixed and labeled for TbG63 and stained with DAPI to visualize the nucleus (N) and kinetoplast (K). Scale bar: 1 µm.

 

Figure 5
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  		Fig. 5. Overexpression of TbG63 affects Golgi morphology. (A) Control cells (first row) or those expressing full-length TbG63 tagged with either YFP or BB2 were fixed and labeled for Golgi (GRASP) or TGN (GRIP), and DNA (DAPI). (B) Cells expressing an N-terminally truncated TbG63 (YFP-TbG63{Delta}N) were fixed and labeled for Golgi (GRASP) and DNA (DAPI). Note that the absence of the N-terminus prevents changes to Golgi morphology. Scale bar: 1 µm. (C) Cryo-iEM of parasites overexpressing full-length YFP-TbG63 visualized using antibodies to GFP followed by 10 nm protein-A-gold. Control, untransfected cell. The three different phenotypes of altered Golgi morphology are arciform, vesiculated and tubulo-vesiculated. Scale bar: 0.25 µm.

 

Figure 6
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  		Fig. 6. Depletion of TbG63 in procyclic parasites. (A) Fluorescence microscopy of cells fixed after mock treatment (left) or after 72 hours induction of RNAi (right), and labeled for TbG63 (top row) or {epsilon}-COP (bottom row). Scale bar: 1 µm. (B) Western blotting using antibodies against TbG63 or {epsilon}-COP.

 

Figure 7
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  		Fig. 7. Depletion of TbG63 in bloodstream parasites inhibits cell growth. (A) Growth curves of control cells and those treated with doxocyclin to induce RNAi. Graph shown is representative of two experiments. (B) Western blotting of cells after 72 hours of induction using antibodies against TbG63 and tubulin as loading control.

 

Figure 8
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  		Fig. 8. VSG export in bloodstream parasites is not affected by depletion of TbG63. Pulse-chase kinetics analysis of VSG arrival at the cell surface. (A) Autoradiograph of pulse-labeled material recovered from soluble (S) and pellet (P) fractions following purification with ConA-Sepharose, from control, non-induced parasites (ND) or parasites induced with doxycyclin for 72 hours to deplete TbG63 (DOX). (B) Western blot of cells used for the VSG export assay, probed with anti-TbG63 antibody to demonstrate depletion of the protein and anti-tubulin as loading control. (C) Quantification of data obtained from A.

 

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© The Company of Biologists Ltd 2008