Rab32 Modulates Apoptosis Onset and Mitochondria-associated Membrane (MAM) Properties

Michael Bui,Susanna Y Gilady,Ross E.B Fitzsimmons,Matthew D Benson,Emily M Lynes,Kevin Gesson,Neal M Alto,Stefan Strack,John D Scott,Thomas Simmen

doi:10.1074/jbc.m110.101584

Abstract

The mitochondria-associated membrane (MAM) has emerged as an endoplasmic reticulum (ER) signaling hub that accommodates ER chaperones, including the lectin calnexin. At the MAM, these chaperones control ER homeostasis but also play a role in the onset of ER stress-mediated apoptosis, likely through the modulation of ER calcium signaling. These opposing roles of MAM-localized chaperones suggest the existence of mechanisms that regulate the composition and the properties of ER membrane domains. Our results now show that the GTPase Rab32 localizes to the ER and mitochondria, and we identify this protein as a regulator of MAM properties. Consistent with such a role, Rab32 modulates ER calcium handling and disrupts the specific enrichment of calnexin on the MAM, while not affecting the ER distribution of protein-disulfide isomerase and mitofusin-2. Furthermore, Rab32 determines the targeting of PKA to mitochondrial and ER membranes and through its overexpression or inactivation increases the phosphorylation of Bad and of Drp1. Through a combination of its functions as a PKA-anchoring protein and a regulator of MAM properties, the activity and expression level of Rab32 determine the speed of apoptosis onset.

Highlights

Mitochondria are in extensive contact with the secretory pathway
When searching for Rab proteins that are candidate regulators of mitochondria-associated membrane (MAM) formation and targeting, we focused on Rab32, because it is thought to be enriched on mitochondria [34]
Consistent with this dual localization to the endoplasmic reticulum (ER) and mitochondria and its identity as an AKAP, we found that Rab32 modulates MAM properties and apoptosis onset

Summary

EXPERIMENTAL PROCEDURES

Antibodies and Reagents—All chemicals were from Sigma, except for H89 (Axxora, San Diego). Western blotting procedures were done according to standard protocols using goat-anti mouse/rabbit secondary antibodies conjugated to Alexafluor 680/750 (Invitrogen) on an Odyssey infrared imaging system (LICOR, Lincoln, NE). Mitochondria were separated from the MAM as follows; HeLa cells were grown to confluency on 15 20-cm dishes and homogenized using a ball-bearing homogenizer as above in 4 ml of isolation buffer (250 mM mannitol, 5 mM HEPES, pH 7.4, 0.5 mM EGTA, 0.1% BSA). We processed HeLa cells for immunofluorescence microscopy and incubated them with antibodies against endogenous Rab. Several spots of Rab staining co-labeled with PDI and MitoTracker (Fig. 1A, white arrowheads). Cols designed to answer the distribution of Rab between the cytosol and ER and mitochondrial membranes (Fig. 1B), on domains of the ER (Fig. 1C), and between mitochondria and the MAM (Fig. 1D). Cium release was triggered by the addition of 200 ␮M histamine, To further examine the localization of Rab, we analyzed its whereas the inhibition of SERCA was achieved with the addi- distribution along the secretory pathway and on domains of tion of 1 ␮M thapsigargin

RESULTS

We incubated HeLa cells with

Findings

DISCUSSION