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

Antibodies and Reagents

All chemicals were from Sigma, except for H89 (Axxora, San Diego). The Rab32 and Rab32 mutant expressing plasmids and the rabbit anti-Rab32 antiserum have been described previously (34). The anti-phospho Drp1 has been described (40). The rabbit anti-calnexin antiserum was generated using a cytosolic peptide by Open Biosystems, Huntsville, AL. Antibodies against GAPDH and MPR46 were provided by Tom Hobman, Edmonton, Alberta, Canada. Antibodies to Sec23 (Abcam, Cambridge, UK), PDI, Drp1, ACAT1 (Thermo, Golden, CO), caspase-3, caspase-8, phospho-Bad serine 112 and 136 (Cell Signaling, Danvers, MA), β-tubulin (Calbiochem), mitochondrial complex 2 (Mitosciences, Eugene, OR), Mitofusin-1 (Abnova, Taipeh, Taiwan), Mitofusin-2 (Sigma), PKA RII (Santa Cruz Biotechnology, Santa Cruz, CA), FLAG tag, Bad, phospho-Bad serine 155 (Millipore, Billerica, MA) were purchased as indicated. HeLa cells were from ECACC (Porton Down, UK) and cultured in DMEM (Invitrogen) containing 10% fetal bovine serum (Invitrogen). Human Rab32 siRNAs (HSS116975) were from Invitrogen. TRAIL was from Millipore (Billerica, MA). The AnnexinV/PI kit was from Calbiochem.

Immunofluorescence Microscopy, Transfections, Western Blotting

Processing for immunofluorescence microscopy was performed as follows. HeLa cells were grown on coverslips for 24 (untransfected cells) or 48 h (when plasmid- or siRNA-transfected). Cells were washed with PBS containing 1 mm CaCl₂ and 0.5 mm MgCl₂ (PBS++) and fixed with 4% paraformaldehyde for 20 min. After washing with PBS++, cells were permeabilized for 1 min with 0.1% Triton X-100, 0.2% BSA in PBS++. Cells were then incubated with primary antibodies (1:100) and secondary antibodies in PBS++, 0.2% BSA for 1 h each, interrupted with three washes using PBS++. All secondary antibodies were AlexaFluor-conjugated 350, 488, or 546 (Invitrogen) used at 1:2,000. Samples were mounted in Prolong AntiFade (Invitrogen). Images were obtained with an Axiocam on an Axio Observer microscope (Carl Zeiss, Jena, Germany) using a 100× plan-Apochromat lens. All images were iteratively deconvolved using the Axiovision 4 software. Deconvolved images were enhanced with Photoshop (Adobe, San Jose, CA) using the levels functions only, until reaching saturation in the most intense areas of the image. Transfections were done using MetafectenePRO (Biontex, Martinsried, Germany), according to the manufacturer's instructions. 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).

Membrane Fractionation, Protein-Protein Binding, and Biotinylation Assays

Mitochondria and light membranes were separated as described previously (12). The MAM OptiPrep^TM gradient protocol was described previously (2). 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). Debris and nuclei were removed by 5 min of centrifugation at 600 × g in 15-ml Corex tubes in a JA-20 rotor. The supernatant was centrifuged at 8,500 rpm in a JA-12 rotor for 10 min to pellet crude mitochondria. Subsequently, microsomes were pelleted at 100,000 × g for 1 h in a TLA120.2 rotor. The previously isolated mitochondria were resuspended in 1 ml of isolation medium and layered on top of 8.5 ml of Percoll isolation medium (225 mm d-mannitol, 25 mm HEPES, pH 7.4, 1 mm EGTA and 20% Percoll (v/v)) in a 10-ml Ultraclear polycarbonate Beckman tube. The tube was centrifuged for 30 min at 30,500 rpm (95,000 × g) in a Ti-70 rotor with slow acceleration and deceleration, after which purified mitochondria (¾ down the tube) and MAM were removed from the Percoll gradient (located above the mitochondria). Percoll was removed from the mitochondrial fraction, and the MAM fraction was diluted 5-fold with fresh isolation medium and re-centrifuged at 60,000 rpm in a TLA120.2 rotor for 1 h. Equal proportional amounts were loaded for all fractions.

Calcium Measurements

ER calcium handling was measured on HeLa cells transfected as indicated and loaded with 2 μm FURA-2 (Invitrogen). 2 × 10⁶ cells were trypsinized and resuspended in DMEM, 10% fetal bovine serum. Cells were pelleted at 800 rpm and resuspended in 2 ml of Tyrode's buffer (2.5 mm KCl, 135 mm NaCl, 10 mm glucose, 1 mm MgCl₂, 1 mm CaCl₂, pH 7.4). Cell suspensions were monitored for light emission at 510 nm after excitation at 340 and 380 nm on an 814 photomultiplier detection system (PTI, Birmingham, NJ). Calcium release was triggered by the addition of 200 μm histamine, whereas the inhibition of SERCA was achieved with the addition of 1 μm thapsigargin.

Rab32 Localizes to the ER and Mitochondria

Rab32 is a small GTPase that is ubiquitous in mammals and whose highest expression levels are found in liver, heart, spleen, and testis (41, 42). In skin, it appears to play a role in melanocyte biogenesis (43, 44). However, its trafficking properties have so far not been addressed in cells that lack melanosomes. Because HeLa cells are a well accepted model system to study intracellular trafficking and apoptosis onset, we decided to investigate the localization and function of Rab32 in these cells. We processed HeLa cells for immunofluorescence microscopy and incubated them with antibodies against endogenous Rab32. We detected a punctate staining pattern that appeared like a composite of ER and mitochondria (Fig. 1A). As a marker for the ER, we used the oxidoreductase PDI, which is found in all domains of the ER (see below). When analyzing the Rab32 staining pattern, we found that Rab32 showed considerable overlap with MitoTracker as published (Fig. 1A, red arrowheads) (34), but it was also found on spots that were exclusively labeled with anti-PDI antibodies (Fig. 1A, blue arrowheads). Interestingly, several spots of Rab32 staining co-labeled with PDI and MitoTracker (Fig. 1A, white arrowheads). To determine which part of the ER was enriched with Rab32, we subjected cellular homogenates to various subcellular fractionation protocols designed to answer the distribution of Rab32 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). First, we separated cellular membranes into cytosol and heavy and light membranes to determine the extent of Rab32 membrane association. Although the majority of endogenous Rab32 was membrane-associated, roughly one-third was found in the cytosol (Fig. 1B). To further examine the localization of Rab32, we analyzed its distribution along the secretory pathway and on domains of the ER using a 10–30% OptiPrep^TM gradient protocol. With this method, we can routinely determine whether an ER protein is found leaking to the late secretory pathway, or resides on the rough ER (rER) or on the MAM; on our Optiprep gradients, endosomal proteins peak at the top of this gradient (MPR46), rER proteins peak in the middle fractions 3–5 (Sec61α), and MAM proteins co-peak with mitochondrial markers at the bottom in fraction 6 (calnexin) (2). To ease detection of rare proteins, we split this gradient into six fractions. This protocol allowed us to detect significant overlap of Rab32 with the endosomal mannose 6-phosphate receptor (MPR46) and cytosolic GAPDH (Fig. 1C) in fractions 1 and 2, but we also noticed considerable amounts of Rab32 co-migrating with the transitional ER marker Sec23 in fraction 4, the rER marker Sec61α in fractions 4 and 5, and the mitochondrial complex 2 in fraction 6 (Fig. 1C). Because the OptiPrep^TM protocol can distinguish between rER and MAM, but not between MAM and mitochondria, we also separated ER and mitochondrial membranes of HeLa cells on a Percoll gradient (1, 45). With this protocol, we detected Rab32 on microsomes, mitochondria, and the MAM (Fig. 1D). Therefore, our immunofluorescence analysis and our three fractionation protocols consistently detect endogenous Rab32 in the cytosol, on mitochondria, and on the MAM of HeLa cells, in addition to lesser amounts on membranes of the late secretory pathway as documented previously for overexpressed GFP-tagged Rab32 (43, 44).

Active and Inactive Rab32 Localize to Distinct Intracellular Membranes

Next, we decided to express and analyze FLAG-tagged Rab32 wild type and Rab32 mutants that preferentially bind to GDP (T39N, dominant-negative) or GTP (Q85L, dominant-active) in HeLa cells, to determine whether it is active or inactive Rab32 that shows preferential association with mitochondria and the MAM. Wild type Rab32 co-localized very nicely with the MAM-enriched ER marker calnexin but also showed some overlap with mitochondria (Fig. 2A). The dominant-active Q85L mutant still co-localized with calnexin, but it showed more cytosolic staining than wild type Rab32 and did not exhibit any significant overlap with mitochondria (Fig. 2B). In contrast, the T39N dominant-negative mutant showed some overlap with both mitochondria and the ER, but it also caused the clustering of mitochondria close to the nucleus, as published previously (Fig. 2C) (34). These results suggested that GTP-bound Q85L and GDP-bound T39N might have opposing distributions in terms of their overlap with mitochondria. To test for that possibility, we first fractionated homogenates of Rab32-transfected cells into cytosol and heavy and light membranes and probed for Rab32 constructs (Fig. 2D). The membrane-associated moiety of Rab32Q85L that fractionated with heavy membranes was significantly lower than for either wild type or T39N Rab32 (46% versus 65 and 71%, respectively). Therefore, this protocol confirmed our immunofluorescence microscopy findings, because heavy membranes contain MAM and mitochondrial markers (Fig. 1B). Using our OptiPrep^TM protocol, we confirmed further that Rab32Q85L preferentially co-fractionates with peripheral membranes, whereas Rab32T39N co-fractionates with perinuclear membranes of the ER and mitochondria (Fig. 2E). The amount of endogenous Rab32 and Rab32T39N found at the bottom of our gradient in fractions 5 and 6 amounted to around 50% of total, whereas only about 35% of overexpressed wild type Rab32 and about 20% of Rab32Q85L associated with these fractions. Given the bipartite localization of Rab32 to the ER and mitochondria and the opposing distributions of GDP and GTP-bound Rab32 along the secretory pathway, we therefore hypothesized that Rab32 could be involved in cargo targeting to the mitochondria or the MAM.

Rab32 Controls the Intracellular PKA Distribution and Signaling

To examine a role of Rab32 in intracellular cargo distribution on the MAM and mitochondria, we chose to first focus on PKA and its substrates, because this kinase signals from mitochondria and is a known interactor of Rab32 (34). Previously, overexpressed GFP-tagged Rab32 had been shown to recruit PKA RIIα to melanosomes, a function that is important for melanosome biogenesis (44). However, because membrane-bound endogenous Rab32 localizes predominantly to the ER and mitochondria in HeLa cells that lack melanosomes (Fig. 1, C and D), we hypothesized that the AKAP function of Rab32 could go beyond melanosome biogenesis and could extend to mitochondria. We first examined the intracellular localization of PKA RIIα in control cells and cells expressing wild type Rab32 and its mutants. We found that increased amounts of Rab32 led to an increased association of PKA with ER membranes that overlapped partially with mitochondria (Fig. 3A). Similarly, the expression of a GDP-bound Rab32 led to the increased association of Rab32 with heavy membranes, concomitant with mitochondrial fusion, typical for this construct (Fig. 3, A and B). This relocation of PKA coincided with increased amounts of PKA on heavy membranes that contain mitochondria and the MAM (Fig. 3B). Conversely, we saw opposite effects with a GTP-bound Rab32 mutant that led to an increased association of PKA with light membranes, and a reduction of PKA on membranes of the MAM and mitochondria (Fig. 3B). The expression of a Rab32 mutant that is not able to bind to PKA resulted in a marked reduction of membrane-bound PKA, in particular on light membranes, and in a relocation of PKA to the cytosol. This was evident by immunofluorescence and fractionation. Overexpression levels versus endogenous Rab32 were in the order of 2–4-fold (Fig. 3C). Together, our results demonstrate that the AKAP characteristics and the GTPase activity of Rab32 determines PKA targeting to heavy membranes and the MAM, leading us to hypothesize that Rab32 alters mitochondrial PKA signaling.

Thus, we first tested this hypothesis by analyzing the individual phosphorylation status of the Bcl2 family protein Bad, given the precedent of AKAP121 regulating the phosphorylation state of Bad on mitochondria (46). Our results show that the overexpression of Rab32 wild type and Rab32 T39N roughly doubled the phosphorylation of Bad on the human equivalent PKA site of mouse Bad serine 155 (serine 118), consistent with their ability to lead to increased targeting of PKA RIIα to heavy membranes (Fig. 3C). Both the activation of Rab32 (Rab32Q85L) and the expression of a Rab32 mutant that is not able to promote PKA RIIα localization to cellular membranes did not affect Bad serine 155 levels but led to decreases in Bad phosphorylation on serine 136 that is a preferred substrate of Akt (Fig. 3C) (47). One explanation for these changes could lie in an alteration of Bad targeting. However, our results show that this was not the case regardless of the activity and expression level of Rab32 (Fig. 3D). Because PKA mediates Bad phosphorylation on serine 155, we also determined the localization of Bad Ser(P)-155 (48). Under all conditions, the amounts of Bad that were phosphorylated on serine 155 were mostly associated with heavy membranes, suggesting that Rab32 does not influence the localization of PKA-phosphorylated Bad (Fig. 3D). Together, our results suggest that Rab32 dictates the extent of Bad PKA phosphorylation on serine 155 but not its localization.

To further investigate the hypothesis that Rab32 modulates PKA signaling on mitochondria and impacts the Bad phosphorylation state, we depleted Rab32 using siRNA. Again, we tested whether Rab32 expression levels affected the localization of PKA RIIα. Contrary to the overexpression of wild type Rab32, we detected a loss of PKA RIIα on heavy membranes and a reduction of the PKA RIIα overlap with mitochondria (Fig. 3E). This effect resulted in reduced Bad phosphorylation on serine 155 (Fig. 3E), but like Rab32 overexpression, Rab32 knockdown does not affect other Bad phosphorylation sites (Fig. 3, B and E). Together, our results demonstrate that Rab32 determines the association of PKA with the MAM and mitochondria and thus modulates Bad phosphorylation on serine 155. In addition, the activation of Rab32 also decreases phosphorylation of Bad on serine 136. We evaluate the effects and consequences of this interference further below and under the “Discussion.”

Next, we aimed to test whether other mitochondrial PKA substrates are also under the influence of Rab32. We chose to analyze dynamin-related protein 1 (Drp1), because the inactivation of this protein by PKA leads to elongation of mitochondria, whereas its activation leads to mitochondrial fragmentation. PKA performs this regulatory function by phosphorylating serine 656 in rat Drp1 (serine 637 of human Drp1); activation of PKA leads to an approximately doubled phosphorylation level of serine 656 in PC12 cells (40, 49). We therefore asked if Rab32 activity also influences the activity of Drp1. An analysis of lysates using a phospho-Drp1-specific antibody showed that this was indeed the case (Fig. 4A). Wild type Rab32 and Rab32T39N increased the Drp1 P656 signal 2- and 2.5-fold, respectively, thus resembling both published Drp1 activation levels and the increases we had observed for phospho-Bad. We could not detect significant changes for the other constructs.

We next analyzed whether the alteration of Drp1 activity coincided with altered Drp1 targeting. Our fractionation protocol that separates heavy and light membranes from the cytosol showed that overexpression of most Rab32 constructs with the exception of Rab32Q85L led to a relative increase of Drp1 on heavy membranes (Fig. 4B). Depletion of Rab32 had no effect. Together, our results showed that the activity of Rab32 can modulate the phosphorylation state and the localization of Drp1.

We next asked whether these observations could explain the clustering of fused mitochondria in a perinuclear area when overexpressing Rab32T39N (Fig. 4C) and to a lesser extent wild type Rab32 (Fig. 3A). To investigate this possibility, we combined the dominant-negative Rab32T39N with the inhibition of PKA. Treatment of transfected cells with the PKA inhibitor H89 led to increased mitochondrial fragmentation, but it was not able to rescue the clustering of mitochondria in the perinuclear area, seen with high expression levels of Rab32T39N (Fig. 4C), suggesting that Rab32 affects both mitochondrial morphology and distribution through distinct mechanisms. We next addressed the putative influence of Rab32 on mitochondrial structure and the activity of Drp1 with HeLa cells depleted of Rab32. Similar to the incubation of cells with H89, we saw an increase of mitochondrial fragmentation when we knocked down Rab32 (Fig. 4D). This fragmentation of mitochondria could be connected to a roughly 30% decrease of Drp1 phosphorylation, known to lead to hyperactive Drp1 and increased mitochondrial fragmentation (data not shown) (40). We therefore demonstrate that Rab32 controls mitochondrial fission by PKA-mediated inactivation of Drp1. Together, our results show that the manipulation of the expression level and activity of Rab32 leads to a profound change of PKA signaling, mediated by the alteration of PKA localization and resulting in altered activities of the PKA downstream targets Bad and Drp1.

Rab32 Dominant-negative and Dominant-active Constructs Influence the Composition of the MAM

Besides a role for the intracellular distribution of PKA, our immunofluorescence and subcellular fractionation studies (Figs. 1 and 2) suggested that Rab32 could also regulate an ER-associated trafficking step or ER tubule fusion mechanism. From the dual localization of Rab32 to the ER and mitochondria, we hypothesized that Rab32 could specifically affect the MAM, similar to what we had previously described for the cytosolic sorting connector protein PACS-2 (2). To examine this possibility, we compared the composition of the MAM of control HeLa cells with the MAM of HeLa cells that overexpress Rab32 mutants or of HeLa cells that have been depleted of Rab32. We focused on the abundant MAM-enriched chaperone calnexin, of which we detect on average close to 70% of the total signal on the MAM and the remaining amount on membranes of the rough ER (2). We analyzed the MAM localization of calnexin first by immunofluorescence. Cells transfected with an empty plasmid showed a partial overlap of the calnexin and mitochondrial staining patterns as published previously by our laboratory (2). In contrast, cells transfected with a dominant-active form of Rab32 showed reduced but not abolished overlap of these two patterns (Fig. 5A). Cells transfected with the dominant-negative Rab32T39N (and to a lesser extent of Rab32 wild type) frequently showed mitochondrial fusion and clustering close to the nucleus (see also Fig. 4C), coinciding with a restriction of calnexin/mitochondrial overlap to the perinuclear area. Next, we aimed to understand if any of the Rab32 constructs resulted in a redistribution of calnexin and other MAM markers within the ER. We chose to answer this question by subcellular fractionation, and we analyzed the intra-ER distribution of calnexin. We also analyzed mitofusin-2, a marker of the MAM (5), and PDI to assay for the overall proximity of the ER with mitochondria. In HeLa cells that were transfected with dominant-active Rab32Q85L, we detected an even distribution of calnexin between heavy membranes and light membranes, indicative of a relocation of calnexin away from the MAM (Fig. 5B). Neither PDI nor mitofusin-2 showed an altered distribution upon Rab32 activation. To further examine this question, we used our OptiPrep^TM protocol, because this protocol, but not the classical Percoll protocol, can assay for localization of ER proteins to various membranes of the secretory pathway (Fig. 5C). Whereas control cells showed less than 40% of the total amount of calnexin on transitional ER and rER membranes, cells that express Rab32Q85L targeted an additional 30% of calnexin to cellular membranes other than the MAM (Fig. 5C). The expression of wild type Rab32 or Rab32T39N had no effect for the calnexin distribution when assayed for either the heavy and light membrane distribution or the OptiPrep^TM distribution (Fig. 5, B and C). We did not detect any increase of the amount of calnexin on the plasma membrane by biotinylation (data not shown), indicating that calnexin is exclusively relocated within the ER upon activation of Rab32. Consistent with the absence of ER or mitochondrial morphology changes, we were unable to detect changes of the localization of PDI, an ER protein that is not enriched on the MAM, or of the mitochondrial MAM marker mitofusin-2, indicating that activation of Rab32 selectively reduces the enrichment of calnexin to the MAM (Fig. 5, B and C).

We next examined whether the knockdown of Rab32 also affects the distribution of any of the aforementioned markers of the ER and the MAM. Both of our fractionation protocols and our immunofluorescence protocol excluded that possibility (Fig. 5, D and E) (data not shown). Our results therefore suggest that the activation status of Rab32 but not its expression levels influence the composition of the MAM.

Activity of Rab32 Modulates Apoptosis Onset and ER Calcium Handling

The roles of Rab32 for PKA localization on mitochondria and for the composition of the MAM implicate Rab32 in the regulation of apoptosis onset. Precedents for these connections are AKAP121, the mitochondrial AKAP that blocks apoptosis onset, and PACS-2, the MAM sorting protein that promotes apoptosis onset (12, 46). We therefore decided to test whether Rab32 similarly plays a role for apoptosis onset. From our results so far, we had determined that Rab32 influences two modifications of Bad known to regulate apoptosis onset; the expression levels of Rab32 influence the PKA-mediated phosphorylation of Bad on serine 155, whereas its activation leads to reduced serine 136 phosphorylation of Bad, known to promote apoptosis. Additionally, Rab32 activation also alters the composition of the MAM that accommodates pro-apoptotic calcium signaling. To further examine the consequences of these observations, we first chose to expose HeLa cells to the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), because TRAIL is known to depend on the formation of the MAM and also on the action of Bad (27, 50).

We incubated HeLa cells with 500 ng/ml TRAIL, an extrinsic inducer of cell death for 4 h. After this incubation period, cells transfected with an empty plasmid or with scrambled siRNA showed ∼20% of cell death when assayed for positive annexin V and propidium iodide staining by flow cytometry (Fig. 6A). Consistent with the Bad serine 136/155 and Drp1 serine 656 phosphorylation patterns observed after interference with Rab32 (Fig. 3), the expression of dominant-active FLAG-tagged Rab32Q85L more than doubled the number of dead or dying cells (Fig. 6A). In contrast, raising the expression levels of Rab32 by transfection of FLAG-tagged wild type Rab32 halved the number of dead or dying cells, as did the expression of dominant-negative FLAG-tagged Rab32T39N (Fig. 6A). Transfection of Rab32L188P or depletion of Rab32 led to an acceleration of apoptosis onset. To confirm and extend these results, we examined the generation of active caspase fragments during TRAIL-induced apoptosis onset in HeLa cells with altered Rab32 expression patterns. We examined early apoptotic events by monitoring the generation of active caspase-8 and late apoptotic events by monitoring generation of active caspase-3. Our results show that the block in apoptosis onset, as seen after Rab32 or Rab32T39N overexpression, coincides with a block in the formation of active caspase-3, but not in a block in the formation of active caspase-8 (Fig. 6B). Analogous to our flow cytometry analysis of Rab32 siRNA-transfected cells, Rab32 silencing coincides with the increased formation of active caspase-3 after treatment with TRAIL (Fig. 6B).

To further characterize the role of Rab32 in apoptosis onset, we next incubated HeLa cells for 24 h with thapsigargin. Overall, the pattern of apoptosis speed in this scenario resembled the one from TRAIL-induced apoptosis, albeit in a less pronounced way (Fig. 6C). Interestingly, when we attempted to recreate a similar effect with the pan-kinase inhibitor staurosporine, we obtained a very different readout, when compared with thapsigargin or TRAIL-induced apoptosis. Although none of the transfections led to protection from apoptosis, the activation of Rab32 (Rab32Q85L) led to a marked increase of apoptosis speed (Fig. 6D). This finding confirms that Rab32Q85L does not regulate apoptosis as an AKAP alone, because staurosporine interferes with the activity of PKA, among other kinases (51).

We therefore hypothesized that the Rab32 levels and activation states influence apoptosis in a combination of its distinct properties as an AKAP and as a regulator of MAM composition (Rab32 Q85L) and ER apposition with mitochondria (Rab32 T39N), which could result in altered ER calcium handling (12). To further investigate this possibility, we incubated cells where we had manipulated Rab32 levels or activities with thapsigargin to increase cytosolic calcium through the inhibition of SERCAs in the presence and absence of the mitochondrial calcium uniport inhibitor Ru386 (52). As shown in Fig. 7, A–C, thapsigargin led to an increase of cytosolic calcium that was potentiated by overexpression of wild type and dominant-negative Rab32 T39N. Interestingly, in the presence of dominant-active Rab32 Q85L, this increase was only observed when we inhibited mitochondrial calcium import (Fig. 7C). Conversely, histamine-induced cytosolic calcium transients do not depend on Rab32, because we could not detect a significantly altered response with either manipulated Rab32 activities or expression levels (Fig. 7D). Together, these results demonstrate that Rab32 regulates apoptosis onset from a complex combination of properties, including its identity as an AKAP, and a regulator of MAM properties and calcium handling.