MICAL-L1 is required for cargo protein delivery to the cell surface.

R Sikora,A Zahraoui,F Niedergang,P Bassereau,M Alqabandi,P Bun,L Danglot,T Galli

doi:10.1242/bio.058008

Abstract

ABSTRACTSecreted proteins are transported along intracellular route from the endoplasmic reticulum through the Golgi before reaching the plasma membrane. Small GTPase Rab and their effectors play a key role in membrane trafficking. Using confocal microscopy, we showed that MICAL-L1 was associated with tubulo-vesicular structures and exhibited a significant colocalization with markers of the Golgi apparatus and recycling endosomes. Super resolution STORM microscopy suggested at the molecular level, a very close association of MICAL-L1 and microdomains in the Golgi cisternae. Using a synchronized secretion assay, we report that the shRNA-mediated depletion of MICAL-L1 impaired the delivery of a subset of cargo proteins to the cell surface. The process of membrane tubulation was monitored in vitro, and we observe that recombinant MICAL-L1-RBD domain may contribute to promote PACSINs-mediated membrane tubulation. Interestingly, two hydrophobic residues at the C-terminus of MICAL-L1 appeared to be important for phosphatidic acid binding, and for association with membrane tubules. Our results reveal a new role for MICAL-L1 in cargo delivery to the plasma membrane.

Highlights

Membrane trafficking is a tightly regulated process required for plasma membrane (PM) homeostasis, adhesion, and cell polarity
MICAL-L1 is associated with Golgi like structure and with cytoplasmic tubulo-vesicles We used an affinity-purified polyclonal antibodies raised against the C-terminal domain to probe an immunoblot of cell extracts prepared from untransfected, Scramble and shRNAs targeting MICAL-L1 transfected HeLa cells
Using our affinity purified antibody, we showed that MICALL1partially colocalized with Golgi apparatus (GA) as well as with recycling endosomes (RE) markers

Summary

Introduction

Membrane trafficking is a tightly regulated process required for plasma membrane (PM) homeostasis, adhesion, and cell polarity. Rab plays a role in the exocytosis of AP-1B-dependent cargo (Ang et al, 2003) It is required for the outgrowth of the primary cilium, and regulates apical protein localization in intestinal cells (Nachury et al, 2007; Sato et al, 2007; Yamamura et al, 2008). Rab regulates tight junction assembly by controlling the delivery of tight junction proteins and downregulation of PKA activity (Köhler et al, 2004; Marzesco and Zahraoui, 2005; Morimoto et al, 2005). It regulates membrane trafficking between GA and PM (Nokes et al, 2008)

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Conclusion