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FEBS Letters 581 (2007) 5765–5768

The heretotrimeric G subunit Gai is present on mitochondria

John S. Lyssand, Sandra M. Bajjalieh* Department of Pharmacology, University of Washington Seattle, Box 357750, WA, USA

Received 18 October 2007; revised 7 November 2007; accepted 12 November 2007

Available online 26 November 2007

Edited by Gianni Cesareni

2. Materials and methods Abstract Receptors that signal through heretotrimeric GTP binding (G) mediate the majority of intercellular com- munication. Recent evidence suggests that receptors acting 2.1. Antibodies Antibodies directed against Ga isoforms were purchased from BD through G proteins also transfer signals across the nuclear mem- Biosciences (Gai, (#612702 generated against an epitope common to brane. Here we present cell fractionation and immunolabeling Gai1–3); Gaq, (#612704); Gas, (#612670)). Anti-RPTPb was pur- data showing that the heretotrimeric subunit Gaiis chased from Transduction Laboratories. Anti-porin antibody was associated with mitochondria. This finding suggests that G pro- from Invitrogen (#A31855) and anti-calreticulin was from BD Biosci- tein signaling may be a feature common to all mem- ences (#612136). The anti-Ga antibodies were tested in Western blots branes. of HEK cell lysates and found to label single bands of the appropriate 2007 Federation of European Biochemical Societies. Published molecular weight (not shown). by Elsevier B.V. All rights reserved. 2.2. Cell fractionation Keywords: ; Mitochondria; G protein HEK293T cells were grown to confluence in 3 · 150 mm plates. Medium was removed and cells were washed 1x with cold PBS. Cells were brought up in 10 mL isolation buffer (5 mM HEPES pH 7.55, 250 mM mannitol, 0.5 mM EGTA & 0.1% BSA) and homogenized with a dounce homogenizer (10 loose passes, then 10 tight). Unbroken cells and nuclei were removed by centrifugation at 1300 · g for 5 min 1. Introduction at 4. The resulting post-nuclear supernatant (PNS) was then centri- fuged at 10300 · g for 10 min at 4 to isolate mitochondria. The result- ing pellet (P2) and supernatant (S2) were collected and the Communication between cells commonly occurs by activa- mitochondria-enriched pellet was re-suspended in 4 mL isolation buffer tion of plasma membrane receptors that act through heretotri- and centrifuged at 10300 · g for 10 min to re-pellet mitochondria. meric GTP-binding (G) proteins. In non-sensory cells, there Washing of the mitochondria-enriched pellet was repeated an addi- are five classes of G protein distinguished by their alpha sub- tional three times. Samples were collected throughout the washing pro- cedure. Protein concentration was determined using the Bradford units, each of which produces a different cellular effect. The dif- assay (Bio-Rad) with BSA as a standard. ferent Ga classes are denoted Gas, Gai, Gao, Gaq/11, and Ga12/13 (reviewed in [1]). 2.3. Western analyses Although the majority of research into G protein-mediated Twenty-five micrograms of each fraction was resolved on 4–20% signaling has focused on across the plasma gradient Tris–HCl gels (Bio-Rad Laboratories) and transferred to membrane, considerable evidence indicates that G protein- nitrocellulose membranes in Tris-glycine transfer buffer with 10% coupled receptors are also present on the nuclear membrane methanol at 100 V for 1 h at 4. Membranes were incubated with blocking buffer (5% milk in phosphate-buffer saline with 0.05% Tween where they mediate signaling by multiple ligands [2–10]. More 20) and then with primary antibodies diluted in blocking buffer (Dilu- recent studies suggest that signaling via G protein-coupled tions: Gai, 1:500; Gas, 1:250; Gaq, 1:1000; calreticulin, 1:2000; porin, receptors may also occur at mitochondrial membranes. For 1:2000; RPTPb, 1:2000). Antibody binding was detected with HRP- example, immunolabeling and pharmacological studies suggest conjugated secondary antibodies reacted with ECL reagent (Pierce that P2Y-like purine receptors, which normally signal through Dura West). ECL luminescence was quantified on a Kodak 440 image station. G proteins, modulate flux across mitochondrial mem- branes in liver cells [11,12]. In addition, an that hydro- 2.4. Immunolabeling lyzes endocannabinoids, which activate G protein coupled To co-label mitochondria and individual Ga isoforms, HEK293T receptors, is enriched in mitochondrial fractions [13]. And cells were plated onto coverslips and allowed to adhere for 24 h. Cells finally, a that generates were rinsed with free media (SFM) and then incubated with [14], a for a family of G protein-coupled receptors MitoTracker Red (Invitrogen, #A7512) diluted 1:10000 in SFM for (reviewed in [15]), localizes to the mitochondria when 15 min at 37 C. Cells were washed 2· with SFM and then fixed w/ 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min. expressed in cultured cells [16]. Despite evidence for both G Cells were rinsed 3· with PBS + 0.1 M glycine and then blocked for protein-coupled receptors and their ligands at the mitochon- 30 min in blocking buffer (2% Normal goat serum, 0.1% BSA, 0.4% dria, however, the presence of heterotrimeric G proteins there saponin in PBS). Primary antibodies diluted in blocking buffer were has not been established. Here we report a survey of mitochon- added to cells and incubated overnight at 4 (Gai was used at 1:100, Gaq and Gas were used at 1:500). Antibody binding was detected with dria for G protein alpha subunits. goat-anti-rabbit secondary antibodies conjugated to AlexaFluor 488 (Invitrogen) diluted 1:1000 in blocking buffer for 1 h at room temper- ature. Cells are then rinsed 3· with PBS and mounted with Fluor- *Corresponding author. Fax: +1 206 685 3822. mount (Southern Biotechnologies Associates) and imaged with a E-mail address: [email protected] (S.M. Bajjalieh). Leica SL Confocal microscope at 63x magnification. Brightness was

0014-5793/$32.00 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2007.11.044 5766 J.S. Lyssand, S.M. Bajjalieh / FEBS Letters 581 (2007) 5765–5768 increased 50% and contrast was increased 25% uniformly in all cells that was identical to that produced by the mitochondrial images. marker MitoTracker, and the two labels co-localized in over- lay images (Fig. 2). On the other hand, antibodies directed against Gaq did not co-localize with MitoTracker. Instead, 3. Results and discussion Gaq appeared to be largely extra-nuclear, suggesting that most of it is in the ER. We note that anti-Gas also co-localized with As part of a study of lipid signaling at the mitochondria, MitoTracker (not shown) whereas it co-fractionated with the we screened mitochondrial fractions for the alpha subunits ER marker calreticulin (Fig. 1), suggesting that it may accu- of heretotrimeric G proteins. To isolate mitochondria, mulate in the mitochondrial-associated subcompartment of homogenates of HEK-293T fibroblasts were subjected to the ER [17]. The apparent low levels of both Gai and Gaq differential centrifugation. Enrichment of mitochondria was at the plasma membrane is consistent with previous observa- assessed by the presence of the mitochondrial protein porin tions of cultured kidney epithelial cells, in which the majority and the absence of the (ER) protein of G alpha subunits were not concentrated in the plasma mem- calreticulin and the receptor protein tryrosine brane [18]. beta (RPTPb), a plasma membrane protein. We found that These results indicate that in HEK293T fibroblasts Gaiis crude mitochondrial fractions contained a significant amount localized to the mitochondria, and thus that signaling through of ER (Fig. 1A, lane P2), but that ER markers were lost Gai may occur in this . These data support the with multiple washing steps. Analyzing fractions by Western hypothesis that and nucleotides regulate mitochondrial blot for the presence of Ga subunits revealed that Gai, but function via Gai-coupled receptors on the mitochondrial mem- not GasorGaq consistently co-fractionated with porin, brane. Potential actions include nucleotide modulation of suggesting that Gai, is present on mitochondria (Fig. 1A mitochondrial membrane conductance and regulation of mito- and B). chondrial fission in a manner similar to that hypothesized for To confirm the fractionation studies, we examined the cellu- Golgi membrane budding (reviewed in [19]). lar location of Ga subunits by immunolabeling. An antibody The presence of G protein-coupled receptors on intracellular directed against Gai produced labeling in cultured HEK293T membranes was commonly thought to reflect regulatory traf-

A PNS S2 P2 S3 P3 P4 P5 P 6

Porin Calreticulin RPTP β

Gi

G s

G q

B 18 14 Calreticulin 10 RPTPβ 6 Gi 2 Gs 2 Gq

1 Normalized Intensity 0 PNS P2 P3 P4 P5 P 6 Fraction

Fig. 1. Gai co-fractionates with the mitochondrial marker porin. (A) Western blot of HEK cell fractions. Twenty-five micrograms of each fraction was resolved on 4–20% SDS–PAGE gels and subjected to Western blotting using antibodies to the indicated protein. Lanes correspond to post- nuclear supernatant (PNS), first 10,300 · g supernatant (S2), mitochondria pellet (P2), supernatant from first mitochondria wash (S3), mitochondria pellet following first wash (P3), mitochondria pellet following second wash (P4), mitochondria pellet following third wash (P5), and mitochondria pellet following fourth wash (P6). (B) Quantification of protein levels across differential centrifugation enrichment of mitochondria. The net band intensity of the indicated protein was divided by the net intensity of porin in the same lane. The ratio obtained for the PNS sample was set at one, and the other fractions expressed as a proportion of the PNS value. Values reflect the mean of two experiments and are representative of a third. Error bars indicate standard error. J.S. Lyssand, S.M. Bajjalieh / FEBS Letters 581 (2007) 5765–5768 5767

Fig. 2. Gai, but not Gaq, co-localizes with the mitochondrial marker MitoTracker. Shown are representative fields from cells labeled with Mitotracker Red and antibodies directed against the indicated Ga isoform (Green). Overlapping labeling appears yellow.

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