AP-3 and Rabip4' coordinately regulate spatial distribution of lysosomes

PLoS One. 2012;7(10):e48142. doi: 10.1371/journal.pone.0048142. Epub 2012 Oct 29.

Abstract

The RUN and FYVE domain proteins rabip4 and rabip4' are encoded by RUFY1 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 RUFY1 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Protein Complex 3 / genetics
  • Adaptor Protein Complex 3 / metabolism*
  • Adaptor Proteins, Signal Transducing
  • Animals
  • Blotting, Western
  • Cell Line
  • Cell Membrane / metabolism
  • Endosomes / metabolism*
  • Fibroblasts / cytology
  • Fibroblasts / metabolism
  • Flow Cytometry
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Intracellular Signaling Peptides and Proteins / genetics
  • Intracellular Signaling Peptides and Proteins / metabolism*
  • Lysosomal-Associated Membrane Protein 1 / metabolism
  • Lysosomes / metabolism*
  • Mice, Mutant Strains
  • Microscopy, Fluorescence
  • Microtubules / metabolism
  • Protein Binding
  • Protein Subunits / genetics
  • Protein Subunits / metabolism
  • RNA Interference
  • Tetraspanin 30 / metabolism
  • Vesicular Transport Proteins / metabolism
  • rab5 GTP-Binding Proteins / metabolism

Substances

  • Adaptor Protein Complex 3
  • Adaptor Proteins, Signal Transducing
  • Intracellular Signaling Peptides and Proteins
  • Lysosomal-Associated Membrane Protein 1
  • Protein Subunits
  • RUFY1 protein, human
  • Tetraspanin 30
  • Vesicular Transport Proteins
  • early endosome antigen 1
  • rab5 GTP-Binding Proteins

Grant support

This work was supported by Executive Unit for Financing Higher Education, Research, Development and Innovation (UEFISCDI) postdoctoral research grant PNII-RU 122/2010 (VI) and a grant of the Dutch Organization for Chemical Research NWO-CW (to PvdS). Core facilities in Utrecht were partly financed by the Netherlands Organization for Medical Research (ZonMW). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.