Tumor Susceptibility Gene 101 (TSG101) Is a Novel Binding-Partner for the Class II Rab11-FIPs

The Rab11-FIPs (Rab11-family interacting proteins; henceforth, FIPs) are a family of Rab11a/Rab11b/Rab25 GTPase effector proteins implicated in an assortment of intracellular trafficking processes. Through proteomic screening, we have identified TSG101 (tumor susceptibility gene 101), a component of the ESCRT-I (endosomal sorting complex required for transport) complex, as a novel FIP4-binding protein, which we find can also bind FIP3. We show that α-helical coiled-coil regions of both TSG101 and FIP4 mediate the interaction with the cognate protein, and that point mutations in the coiled-coil regions of both TSG101 and FIP4 abrogate the interaction. We find that expression of TSG101 and FIP4 mutants cause cytokinesis defects, but that the TSG101-FIP4 interaction is not required for localisation of TSG101 to the midbody/Flemming body during abscission. Together, these data suggest functional overlap between Rab11-controlled processes and components of the ESCRT pathway

FIP4 localises to the midbody of dividing cells independently of TSG101.

<p>HeLa cells were transfected with constructs encoding the indicated proteins. At 16–18 hours post-transfection, cells were processed for immunofluorescence microscopy and immunostained for α-tubulin. DAPI was used to visualise the nuclei. Images were acquired by confocal microscopy. Scale bar indicates 10 µm. Data are typical of at least three independent experiments.</p

An extensive coiled-coil region of FIP4 mediates the interaction with TSG101.

<p>(<b><i>A</i></b>) Plot depicting the probability of α-helical coiled-coil structure formation in FIP4 as determined using the <i>PairCoil</i> algorithm. (<b><i>B</i></b>) Schematic representation of the FIP4 truncation and point mutants that were tested for TSG101-binding ability. The outcome of the yeast two-hybrid experiments performed (part <i>C</i>) are indicated adjacent to the relevant mutant in the schematic. (<b><i>C</i></b>) Yeast two-hybrid analysis of the interaction between the indicated proteins. Protein-protein interactions were determined by the ability of the transformed yeast to grow on minimal medium lacking tryptophan, leucine and histidine (<i>His−</i>). <i>EV</i>, empty vector. Data are typical of at least three independent experiments.</p

TSG101 localises to the Flemming body during abscission independently of the class II FIPs.

<p>(<b><i>A</i></b> and <b><i>B</i></b>) HeLa cells were transfected with constructs encoding the indicated proteins. At 16–18 hours post-transfection, cells were processed for immunofluorescence microscopy and, where indicated, immunostained for α-tubulin. DAPI was used to visualise the nuclei. Images, from cells expressing relatively low levels of the TSG101 fusion protein, were acquired by confocal microscopy. Scale bar indicates 10 µm. Data are typical of at least three independent experiments.</p

The coiled-coil region of TSG101 mediates the interaction with FIP4.

<p>(<b><i>A</i></b>) Plot depicting the probability of α-helical coiled-coil structure formation in TSG101 as determined using the <i>PairCoil</i> algorithm. (<b><i>B</i></b>) Schematic representation of the TSG101 truncation and point mutants that were tested for FIP4-binding ability. The outcome of the yeast two-hybrid experiments performed (part <i>C</i>) are indicated adjacent to the corresponding mutant in the schematic. (<b><i>C</i></b>) Yeast two-hybrid analysis of the interaction between the indicated proteins. Protein-protein interactions were determined by the ability of the transformed yeast to grow on minimal medium lacking tryptophan, leucine and histidine (<i>His−</i>). <i>EV</i>, empty vector. Data are typical of at least three independent experiments.</p

Expression of TSG101 and FIP4 dominant-negative mutants cause abscission failure.

<p>HeLa cells were transfected with constructs encoding the indicated proteins. At 36–40 hours post-transfection, cells were processed for immunofluorescence microscopy, immunostained for α-tubulin and their nuclei fluorescently-labelled with DAPI. A minimum of 150 transfected cells per experiment were counted and scored for multinucleation (>1 nucleus). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032030#s2" target="_blank">Results</a>, from three independent experiments, are expressed as the mean percentages ± S.D. Statistical significance was determined using an unpaired <i>t</i> test to investigate: (<b><i>A</i></b>) the difference between empty vector and GFP-fusion means, (<b><i>B</i></b>) the difference between GFP-TSG101 and GFP-TSG101 point mutant means and (<b><i>C</i></b>) the difference between GFP-FIP4 and GFP-FIP4 point mutant means. Statistical significance, *p<0.05, **p<0.02. <i>NT</i>, non-transfected; <i>EV</i>, empty vector.</p

TSG101 binds the class II FIPs.

<p>(<b><i>A</i></b>) Yeast two-hybrid analysis of the interaction between the indicated proteins. Protein-protein interactions were determined by the ability of the transformed yeast to grow on minimal medium lacking tryptophan, leucine and histidine (<i>His−</i>). <i>EV</i>, empty vector. (<b><i>B</i></b>) Co-immunoprecipitation analysis of the ability of Xpress-FIPs to co-immunoprecipitate GFP-TSG101 in HeLa cells using an anti-Xpress antibody (<i>SM</i>, starting material; <i>IP</i>, immunoprecipitate). Co-immunoprecipitated proteins were revealed using an anti-GFP antibody. GFP-empty vector (<i>EV</i>) was used as a control. SM load was 3.33%. (<b><i>C</i></b> and <b><i>D</i></b>) HeLa cells were transfected with constructs encoding the indicated proteins. At 16–18 hours post-transfection, cells were processed for immunofluorescence microscopy and immunostained with an anti-Xpress antibody. Cells expressing relatively low levels of the GFP-TSG101 protein are shown in <i>D</i>. DAPI was used to visualise the nuclei. Images were acquired by confocal microscopy. Insets illustrate the midbody region of dividing cells at 2.5× higher magnification. Scale bar indicates 10 µm. Data are typical of at least three independent experiments.</p

Rab11 family expression in the human placenta: Localization at the maternal-fetal interface.

Rab proteins are a family of small GTPases involved in a variety of cellular processes. The Rab11 subfamily in particular directs key steps of intracellular functions involving vesicle trafficking of the endosomal recycling pathway. This Rab subfamily works through a series of effector proteins including the Rab11-FIPs (Rab11 Family-Interacting Proteins). While the Rab11 subfamily has been well characterized at the cellular level, its function within human organ systems is still being explored. In an effort to further study these proteins, we conducted a preliminary investigation of a subgroup of endosomal Rab proteins in a range of human cell lines by Western blotting. The results from this analysis indicated that Rab11a, Rab11c(Rab25) and Rab14 were expressed in a wide range of cell lines, including the human placental trophoblastic BeWo cell line. These findings encouraged us to further analyse the localization of these Rabs and their common effector protein, the Rab Coupling Protein (RCP), by immunofluorescence microscopy and to extend this work to normal human placental tissue. The placenta is a highly active exchange interface, facilitating transfer between mother and fetus during pregnancy. As Rab11 proteins are closely involved in transcytosis we hypothesized that the placenta would be an interesting human tissue model system for Rab investigation. By immunofluorescence microscopy, Rab11a, Rab11c(Rab25), Rab14 as well as their common FIP effector RCP showed prominent expression in the placental cell lines. We also identified the expression of these proteins in human placental lysates by Western blot analysis. Further, via fluorescent immunohistochemistry, we noted abundant localization of these proteins within key functional areas of primary human placental tissues, namely the outer syncytial layer of placental villous tissue and the endothelia of fetal blood vessels. Overall these findings highlight the expression of the Rab11 family within the human placenta, with novel localization at the maternal-fetal interface

Comparative localization patterns of Rab11a and Rab14 in human cell lines.

<p>Rab11a and Rab14 localization was examined in cell lines H1299 (a,d,g,j), HeLa (b,e,h,k) and BeWo (c,f,i,l) by confocal immunofluorescence microscopy. Images a-c, g-i are representative fields of the indicated cells; images d-f, j-l are zoomed images. Ten micron scale bars included on each image.</p

Co-localization of Rab11 subfamily with RCP in human placental villi.

<p>Double immunofluorescence microscopy of placental sample cohort for co-labeling of RCP with (A) Rab11, (B) Rab14, and (C) Rab25. Red label: Rabs, Green label: RCP, Blue label: DAPI nuclear stain). Representative individual staining shown on left with merged image on right. Inset outlined by white box shows magnified area of Rab/RCP co-localization as indicated by yellow color change. Twenty-five micron scale bars included on lower right of each image.</p