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

Viorica Ivan,Livia E Sima,Viola Oorschot,Peter Van Der Sluijs,Judith Klumperman,Emma Martinez-Sanchez,Stefana M Petrescu,Ruben Claudio Aguilar

doi:10.1371/journal.pone.0048142

Viorica Ivan, Livia E Sima + Show 6 more

Open Access

https://doi.org/10.1371/journal.pone.0048142

Copy DOI

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.

Highlights

Lysosomes are dynamic membrane-bound organelles that degrade macromolecules from the endocytic, secretory, and autophagic pathways [1,2]
Lysosomes were traditionally appreciated for their degradative function, but it is clear that they serve more complex roles like plasma membrane repair, and as intracellular signaling platforms [1,2,3]
As expected from the binding data obtained with brain, we found that adaptor protein complexes (AP)-1 from rescued mocha cells did not interact with GST-rabip4’(aa 299–708)

Summary

Introduction

Lysosomes are dynamic membrane-bound organelles that degrade macromolecules from the endocytic, secretory, and autophagic pathways [1,2]. The small GTPse Arl and PLEKHM2 are needed for kinesin-1 accumulation on lysosomes and distribution of lysosomes in the cell periphery [14] Membrane proteins reach their steady state distribution via transport carriers that shuttle cargo between organelles. AP-1 to AP-5, are presently known [15] Their localization to distinct intracellular membrane domains is an important factor in establishing specificity in the formation of transport carriers. Rab GTPases are important regulators of endo-lysosomal transport [23] They recruit effectors to relay the GTPase switch to downstream biological processes and in doing so create membrane heterogeneity in the endosomal network. Such microdomains serve as platforms for the different transport and signaling pathways [24]. We here characterize the interaction between rabip4’ and AP-3 and document for the first time a role for both AP-3 and rabip4’ in the intracellular distribution of lysosomes via a pathway that is downstream of rab

Results

Discussion

Experimental Procedures