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First published online 12 February 2008
doi: 10.1242/jcs.015610

Journal of Cell Science 121, 717-723 (2008)
Published by The Company of Biologists 2008
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A novel HSP90 chaperone complex regulates intracellular vesicle transport

Gregor P. Lotz1,*, Alexander Brychzy2, Stefan Heinz3 and Wolfgang M. J. Obermann1,4,{ddagger}

1 Protein Folding Group, Institute for Genetics, University of Bonn, Römerstr. 164, 53117 Bonn, Germany
2 Cellular Biochemistry, Max Planck Institute of Biochemistry, Am Klopferspitz 18a, 82152 Martinsried, Germany
3 Institute for Transfusion Medicine and Immunohematology, Red Cross Blood Donor Service Baden-Württemberg/Hessen, Sandhofstr. 1, 60528 Frankfurt, Germany
4 Department of Neuroscience and Cell Biology, University of Texas Medical Branch, 301 University Boulevard, Galveston TX 77555, USA


Figure 1
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  		Fig. 1. The co-chaperone TPR1 binds to HSP90 and VAP-33. (A) Representation of the HSP90, TPR1 and VAP-33 constructs used for the two-hybrid experiments. The top row shows, from left to right, full-length TPR1, Tpr1unique and the fragment containing the TPR domain. The column on the left shows, from top to bottom, Hsp90C, full-length VAP-33, VAP-33 lacking its membrane anchor tail, the MSP domain of VAP-33 and the fragment containing the coiled-coil region (represented by blue double-tilde). The plus (+) and minus (–) symbols indicate the presence or absence of an interaction when various constructs were co-transformed in yeast cells and tested for growth as shown in panel B. (B) Constructs in the pAS2-1 vector were co-transformed with pACT2 constructs into S. cerevisiae Y190 as indicated, with empty vectors serving as a control. After growth on SD/–Trp, –Leu plates, cells were re-plated on the same medium to confirm the presence of the pAS2-1 and pACT vectors (left panel) and on SD/–Trp, –Leu, –His plates containing 25 mM 3-amino-1,2,4-triazole (right panel) to select for protein interactions. TPR1 and Tpr1TPR interacted with Hsp90C, whereas TPR1 and Tpr1unique interacted with VAP-33, VAP-33(–tail) and VAP-33MSP. (C) Illustration of the complex comprising HSP90, TPR1 and the membrane-anchored protein VAP-33 based on our yeast two-hybrid data.

 

Figure 2
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  		Fig. 2. Analysis of the TPR1 interaction with HSP90 and VAP-33 by gel filtration chromatography and surface plasmon resonance. (A) Purified HSP90, TPR1 and VAP-33, lacking its membrane anchor, were incubated as indicated in the presence or absence of ATP{gamma}S, separated by gel filtration chromatography on a Superose 6 column and fractions analyzed by SDS-PAGE. Elution profiles of marker proteins are shown on top (thyroglobulin, 669 kDa; ferritin, 440 kDa; catalase 232 kDa; bovine serum albumin, 67 kDa). *, a degradation product of the HSP90 preparation. (B) Tpr1unique binds to VAP-33MSP. Tpr1unique was incubated with either VAP-33 lacking its membrane anchor, or VAP-33MSP, and the mixtures were run on a Superdex 75 column. Co-migration indicated complex formation between Tpr1unique and VAP-33 or VAP-33MSP. Marker proteins are shown on top (bovine serum albumin, 67 kDa; ribonuclease A, 14 kDa). (C) 12-mer peptides containing the C-terminal sequence of HSP90 (90C-12) were covalently coupled to a BiaCore chip. The binding kinetics of TPR1 in the concentration range of 0.1 to 60 µM were tested, and the relative response units during the equilibrium phase of binding to 90C-12 were plotted against TPR1 concentrations. (D) Increasing concentrations (0.1 to 100 µM) of 90C-12 in solution were used to compete for binding of TPR1 to immobilized 90C-12. Binding of TPR1 to HSP90 peptides could be competed using free 12mer peptides from HSP90 (90C-12) but not the control peptide SKL. (E) Cow brain lysate was incubated with or without an HSP90-specific antibody. After pulldown with ProteinG beads, immunoprecipitates were analyzed with antibodies against HSP90 and VAP-33. Antibody reactivity using 1% or 10% of the input (brain lysate) is shown as a standard.

 

Figure 3
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  		Fig. 3. TPR1 downregulation obstructs vesicular transport of the VSVGts045-GFP cargo protein in vivo. (A) Decrease of TPR1 expression levels in HEK293 cells by specific siRNA. Cells were mock transfected, treated with siRNA targeting human TPR1 or with mutated or control siRNA. Equal protein loading is shown by an antibody specific for GAPDH. (B) Time-course of intracellular VSVGts045-GFP protein trafficking in cells treated with control siRNA or siRNA targeted against human TPR1. Localization of VSVGts045-GFP and the Golgi marker GM130 was assessed after 0, 15 and 60 minutes. Bar, 10 µm. (C) Resistance of VSVGts045-GFP protein against EndoHf digestion in cells treated with control siRNA or siRNA specific for TPR1 after 0, 15 and 60 minutes at 32°C. The markers `low' and `high' refer to the endoplasmic reticulum form of VSVGts045-GFP from cells that have been kept at 40°C after and before EndoHf treatment (Balch and Keller, 1986Go). Asterisks indicate two EndoHf-resistant bands, a hallmark of arrival in the medial-Golgi compartment (Schwaninger et al., 1992Go). Presented is a typical result of an experiment performed three times. (D) EndoHf resistancy of VSVG after 0, 15 and 60 minutes in cells treated with control siRNA or siRNA specific for TPR1. Quantification was based on the areas in each blot that correspond to the two bands marked with asterisks in C and is represented as a percentage of the total signal measured. Bars, ± s.e.m.

 

Figure 4
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  		Fig. 4. Transport of the VSVG protein in a cell-free system is susceptible to HSP90-specific drugs and dependent on the interaction between the HSP90-TPR1 chaperone complex and VAP-33. (A) Intra-Golgi transport of VSVG protein is inhibited by geldanamycin, radicicol and macbecin compared with control assays. (B) Intra-Golgi transport can be inhibited by addition of the Tpr1unique fragment but not by bovine serum albumin (BSA) serving as a control. Quantified data are averages of three independent experiments. cpm, counts per minute; GlcNAc, N-acetylglucosamine. Bars, ± s.e.m.

 

Figure 5
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  		Fig. 5. The TPR1-specific antibody mAb292-200 inhibits intra-Golgi transport. 150 ng of antibody against TPR1 (mAb292-200) or an unrelated antibody against the Myc-tag (clone 9E10) serving as a control were added to 50 µl of the transport assay mixture and the transport reaction run for 1 hour, as described in Materials and Methods. The antibody against TPR1 but not the antibody against Myc results in decreased VSVG transport. Bars, ± s.e.m.

 

Figure 6
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  		Fig. 6. TPR1 coprecipitates contain Rab8. TPR1 was isolated from cell extracts using the mAb292-200 monoclonal antibody against TPR1 coupled to beads. Bound material was separated by SDS-PAGE and analyzed with the antibodies indicated and compared with control (ctrl) beads.

 

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© The Company of Biologists Ltd 2008