HCMV infection after knock-down of Rab27a.

<p>(A) Mewo cells expressing non-target control and Rab27a shRNAs were lysed, and Rab27a expression was determined by Western blot. Actin served as loading control. Molecular weights in kDa are indicated. (B–D) Infectious viruses produced in BJ1 Rab27a shRNAs expressing cells. BJ1 untransduced, non-target control and Rab27a shRNAs expressing cells were infected with RCMV288 at a moi of 0.5. After 3 days a fraction of the cells was analysed by flow cytometry to assess the number of infected cells by GFP expression (B). Some cells were lysed at 4 dpi, and expression of Rab27a, actin and HCMV was analyzed by Western blot (C). At 5 dpi, supernatants and the remainder of the cells were harvested, and the number of extracellular and cell-associated infectious viruses was determine on fresh BJ1 cells as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015318#s2" target="_blank">Materials and Methods</a> (D). Data are means plus standard deviation (n = 3). ***, <i>P</i><0.001. MFI: mean fluorescence intensity. IP: infectious particles.</p

Immuno EM localisation of Rab27a on the viral envelope of isolated HCMV viral particles.

<p>Isolated viral particles from BJ1 YFP-Rab27a cells HCMV infected for 5 days were permeabilised with saponin and labelled with antibodies against YFP (A) and HCMV tegument viral protein pp28 (B), and 10 nm protein-A gold. (C) General morphology of HCMV virions by negative staining with 2% uranyl acetate. When viral particles were partially disrupted, uranyl acetate revealed the nucleocapsids. Scale bar, 50 nm.</p

Immuno EM localisation of Rab27a in HCMV-infected cells.

<p>(A and B) Cryosections of BJ1 YFP-Rab27a cells HCMV infected for 5 days at a moi of 3 were labelled for YFP with 10 nm gold-conjugated secondary antibodies. Gold particles were seen over the membrane of vacuoles, small vesicles and tubules, as well as the envelope of virions and dense bodies (black arrowhead), and the vacuole containing them (white arrowhead). N, nucleus; VL, vacuole; V, virion; D, dense bodies. Scale bars, 200 nm.</p

MCMV infection in Rab27a-deficient melanocytes.

<p>M<i>elan-a</i> and <i>ashen-3</i> melanocytes were infected with MCMV, and after 24 h some cells were fixed and MCMV IE1 protein expression analysed by immunofluorescence microscopy (magenta in upper panels). At 48 hpi, supernatants and cells were harvested, and the number of extracellular and cell-associated infectious viruses were determined by plaque assay (histograms). Data are means plus standard deviations (n = 2). ***, P<0.001. PFU: plaque forming units.</p

Expression and localisation of Rab27a in HCMV-infected cells.

<p>(A) Rab27a protein expression during HCMV infection. Equal number of BJ1 cells mock infected or infected with HCMV at a moi of 3 were lysed at the indicated times. Rab27a was analyzed by Western blot where actin served as loading control Autoradiography films were scanned; images were cropped and assembled with Adobe Photoshop. Molecular weights in kDa are indicated. (B) Subcellular localisation of Rab27a in HCMV-infected cells. BJ1 and BJ1-YFP-Rab27a cells were either mock infected (left panels) or infected with HCMV at a moi of 0.5 (right panels). After 5dpi, cells were fixed, permeabilised and stained with anti-Rab27a (green in upper panels) and anti-HCMV glycoprotein gH (red) antibodies. In BJ1-YFP-Rab27a cells, YFP was directly imaged. DNA was stained with DAPI (blue). Dashed lines show the outline of the cells. Scale bars, 20 µm.</p

Rabip4’ localization does not require AP-3.

<p>VSVG-rabip4’-expressing HeLa cells were treated with 5 µg/ml BFA for 15 min at 37°C and stained with a rabbit antibody against rabip4’ and mouse anti-δ-adaptin or mouse anti-TfR, followed by Alexa568-anti-rabbit and Alexa488-anti-mouse IgG. Rabip4’ overexpression did not affect AP-3 sensitivity to BFA. Lower row represents insets of boxed areas. Arrows point to BFA-induced tubulation of rabip4’ and TfR (<b>A</b>). Rabip4s-directed siRNA oligos were transfected in HeLa cells for 3 days. AP-3 distribution was similar in both siRNA-transfected and control cells. Scale bar is 10 µm. Silencing was monitored by Western blotting and gave routinely 80–85% reduction of both rabip4’ and rabip4 isoforms (<b>B</b>). AP-3 siRNA oligos were transfected in HeLa cells for 3 days. Two days after siRNA treatment, cells were transfected with VSVG-rabip4’ for another day and labeled for δ-adaptin and rabip4’. Rabip4’ distribution did not depend on AP-3. Scale bar is 10 µm. Western blots were probed with antibodies against δ-adaptin. The level of δ-adaptin in siRNA-transfected cells was quantified and expressed as % of control (<b>C</b>).</p

Rabip4’, EEA1, and Hrs partially colocalize on endosomes.

<p>HeLa cells were labeled for endogenous rabip4s and EEA1. Arrows denote co-distribution of the two FYVE domain-proteins. Bottom row represents a blow up of indicated areas. The dashed line marks the contour of cells. Scale bar is 10 µm (<b>A</b>). Ultrathin cryosections of HeLa cells expressing VSVG-rabip4’ were immunogold labeled with polyclonal anti-VSVG (10 nm gold particles). VSVG-rabip4’ localized to the endosomal vacuole (E), as well as to surrounding tubular-vesicular membrane profiles characteristic for recycling tubules (arrows). P is plasma membrane. Bar, 200 nm (<b>B</b>). HeLa cells transfected with GFP-rabip4’ and HA-Hrs were labeled for EEA1 (blue) and HA-Hrs (red), while rabip4’ was visualized by EGFP fluorescence. Bottom row represents an enlargement of the indicated areas. Arrows point to endosomes that contain rabip4’, EEA1, and Hrs, and arrowheads indicate endosomes devoid of rabip4’. Scale bar is 10 µm (<b>C</b>).</p

Rabip4’ and AP-3 cooperate in lysosome positioning.

<p>HEK293T cells were depleted of rabip4s or AP-3 and then labeled for immunofluorescence with mouse antibodies against CD63 and TfR (green) or with rabbit antisera specific for cathepsin D and Ti-VAMP (red) (<b>A</b>). HEK293T cells were processed as above and co-labeled with anti-cathepsin D (red) and anti-tubulin (green) antibodies or with anti-cathepsin D antibody (red) and Alexa-488-conjugated phalloidin for actin staining. Nuclei were stained with DAPI. (<b>B</b>). Images represent projections of confocal Z-stacks. Scale bar, 10 µm. Depletion of rabip4s and AP-3 selectively redistributed the lysosomal markers CD63, Cathepsin D, and Ti-VAMP to cellular protrusions (arrows).</p

Rabip4’ interacts specifically and directly with AP-3.

<p>Immobilized GST-rabip4’ (aa 299–708) was incubated with brain cytosol. Bound proteins were resolved by SDS-PAGE and analyzed by tandem mass spectrometry, which yielded β3-adaptin as binding partner (<b>A</b>). Eluates were probed with antibodies against subunits of adaptor complexes showing specificity for AP-3 (<b>B</b>). GST-rabip4’ beads were incubated with detergent extracts from rescued <i>mocha</i> cells and bound proteins were analyzed by Western blot for the indicated AP-3 and AP-1 subunits. The ubiquitous AP-3 specifically interacted with rabip4’ (<b>C</b>). AP-3 was immunoprecipitated from lysates of HeLa cells expressing VSVG-rabip4’ and analyzed by Western blot with antibodies against VSVG and AP-3 subunits. Immunoprecipitation of AP-1 or a monoclonal antibody against HA (control IgG) did not co-immunoprecipitate VSVG-rabip4’ (<b>D</b>). GST-rabip4’ was immobilized on GSH beads and incubated with ³⁵S-labeled AP-3 subunits. Rabip4’ interacted directly with AP-3 through the β3 subunit (<b>E</b>).</p

AP-3 and Rabip4’ Coordinately Regulate Spatial Distribution of Lysosomes

<div><p>The RUN and FYVE domain proteins rabip4 and rabip4’ are encoded by <em>RUFY1</em> and differ in a 108 amino acid N-terminal extension in rabip4’. Their identical C terminus binds rab5 and rab4, but the function of rabip4s is incompletely understood. We here found that silencing <em>RUFY1</em> gene products promoted outgrowth of plasma membrane protrusions, and polarized distribution and clustering of lysosomes at their tips. An interactor screen for proteins that function together with rabip4’ yielded the adaptor protein complex AP-3, of which the hinge region in the β3 subunit bound directly to the FYVE domain of rabip4’. Rabip4’ colocalized with AP-3 on a tubular subdomain of early endosomes and the extent of colocalization was increased by a dominant negative rab4 mutant. Knock-down of AP-3 had an ever more dramatic effect and caused accumulation of lysosomes in protrusions at the plasma membrane. The most peripheral lysosomes were localized beyond microtubules, within the cortical actin network. Our results uncover a novel function for AP-3 and rabip4’ in regulating lysosome positioning through an interorganellar pathway.</p> </div