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. 2015 Sep 1:68:3.27.1-3.27.33.
doi: 10.1002/0471143030.cb0327s68.

Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria-Associated Membrane and Detergent Resistant Membrane Fractions from Transfected Cells and from Human Cytomegalovirus-Infected Primary Fibroblasts

Chad D Williamson  1   2 , Daniel S Wong  3 , Petros Bozidis  4 , Aiping Zhang  1 , Anamaris M Colberg-Poley  1   5   6   7
Affiliations

Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria-Associated Membrane and Detergent Resistant Membrane Fractions from Transfected Cells and from Human Cytomegalovirus-Infected Primary Fibroblasts

Chad D Williamson et al. Curr Protoc Cell Biol. .

Abstract

Increasingly mechanistic virology studies require dependable and sensitive methods for isolating purified organelles containing functional cellular sub-domains. The mitochondrial network is, in part, closely apposed to the endoplasmic reticulum (ER). The mitochondria-associated membrane (MAM) fraction provides direct physical contact between the ER and mitochondria. Characterization of the dual localization and trafficking of human cytomegalovirus (HCMV) UL37 proteins required establishing protocols in which the ER and mitochondria could be reliably separated. Because of its documented role in lipid and ceramide transfer from the ER to mitochondria, a method to purify MAM from infected cells was also developed. Two robust procedures were developed to efficiently isolate mitochondria, ER, and MAM fractions while providing substantial protein yields from HCMV-infected primary fibroblasts and from transfected HeLa cells. Furthermore, this unit includes protocols for isolation of detergent resistant membranes from subcellular fractions as well as techniques that allow visualization of the mitochondrial network disruption that occurs in permissively infected cells by their optimal resolution in Percoll gradients.

Keywords: ER; HCMV; MAM; Percoll gradient; differential centrifugation; human fibroblasts; mitochondria; protein localization; subcellular fractionation; sucrose gradient.

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Figures

Figure 3.27.1
Figure 3.27.1
A flow chart for Basic Protocol 1 is shown. Basic Protocol 1, step 14, separates crude ER (supernatant) from crude mitochondria (pellet). Subsequent steps are grouped by the organelle which is to be purified for clarity and to provide a sense of continuity. To streamline the timing of the procedure and to reduce protein degradation; however, ER and mitochondrial purification steps should be carried out simultaneously.
Figure 3.27.2
Figure 3.27.2
Representative pictures of visible bands seen upon ultracentrifugation as described in Basic Protocol 1. The ER and mitochondria bands are indicated on their respective gradients following ultracentrifugation.
Figure 3.27.3
Figure 3.27.3
Schematic representation of Basic Protocol 2. Critical steps in the procedure, which improve purity of the microsomes, mitochondria, and MAM, are emphasized.
Figure 3.27.4
Figure 3.27.4
Separation of MAM and mitochondrial fractions using self-generating Percoll (30%) gradients. Crude mitochondrial extracts from uninfected or HCMV-infected HFFs were subjected to ultracentrifugation in a Percoll gradient (see Basic Protocol 2). The diagram on the left illustrates the distinct features of banded organelles within the Percoll gradient. Fractions 2 and 3 were collected to isolate purified MAM and mitochondria, respectively. Gradients on the right demonstrate proper banding of MAM and mitochondria in Percoll gradients, as well as highlight the alterations in mitochondrial banding observed during HCMV infection.
Figure 3.27.5
Figure 3.27.5
Western analyses of ER (DPM1), mitochondrial (Grp75, COXII), and MAM (FACL4) markers in HeLa cells lysed by different methods. HeLa cells were fractionated using discontinuous sucrose gradients (see Basic Protocol 1) as detailed in Table 3.27.1. Briefly, cells were lysed using sonication (three times, 5, 10, or 15 sec each), Dounce homogenization (ten strokes), or freeze/thaw cycles (three times 1 hr at −80°C, followed by rapid thawing). Twenty micrograms of total, ER, and mitochondrial protein fractions from each sonication condition were subjected to SDS-PAGE in 10% Bis-Tris NuPage gels (Invitrogen). For homogenization and freeze/thaw lysis conditions, 40 μl of each fraction were used (due to low protein concentrations). Proteins were then transferred to nitrocellulose membranes and probed for DPM1 (1:100 goat anti-DPM1, 1:2000 donkey anti-goat HRP). Membranes were stripped and reprobed for Grp75 (1:2000 mouse anti-Grp75, 1:2500 goat anti-mouse). Similarly, membranes were then stripped and reprobed sequentially for FACL4 (1:250 rabbit anti-FACL4, 1:3000 goat anti-rabbit HRP) then COX II (1:200 goat anti-COXII, 1:2500 donkey anti-goat HRP).
Figure 3.27.6
Figure 3.27.6
Western analyses of fractionated HeLa cells that stably express mEGFP-huPSS-1 fusion protein. Stably transfected cells were fractionated according to the procedure described in Basic Protocol 2 and subcellular fractions were isolated. Fractionated proteins (10 μg) were separated by 10% SDS PAGE and transferred onto nitrocellulose membranes using a semi-dry protein transfer apparatus (BioRad). Blotted proteins were probed against markers for microsomes (anti-DPM1, 1:100 or anti-calreticulin, 1:1000), MAM (mEGFP-huPSS-1 using anti-GFP, 1:100), and mitochondria (anti-COX, 1:100 or anti-Grp75, 1:2500) and with the corresponding horseradish peroxidase-conjugated secondary Ab (1:2000). Reactivity was detected using the chemiluminescent method (Amersham, GE Healthcare).
Figure 3.27.7
Figure 3.27.7
Flotation of detergent-resistant MAM membranes from crude ER or mitochondrial fractions (Protocol 3). A. Crude ER or mitochondria were solubilized with MBST buffer and floated through sucrose gradients (as in Basic Protocol 3). Banding of DRMs in discontinuous sucrose gradients is demonstrated on the right. DRMs band in sucrose between 20–15%. To maximize separation from bulk membranes, it is preferable to band DRMs near the top of the sucrose gradient. Basic protocol 3 utilizes a 30% sucrose buffer cushion, as shown in the diagram to the left. A 20% cushion also allows DRMs to band similarly, while increasing the separation from bulk membranes. B. DRMs from ER/MAM and mitochondria from HCMV infected cells. HFFs were HCMV (strain BADwt)-infected (multiplicity of infection of 3) and ER/MAM and mitochondria were isolated at 48 hrs. Lipid rafts were then isolated from the indicated purified subcellular fractions by cold 1% Triton X-100 extraction and flotation in 5%, 35% and 45% sucrose gradients. Each gradient fraction (20 μl) was resolved by SDS-PAGE and analyzed by Western blots using anti-vMIA/UL37x1 antibody. C. DRMs from ER/MAM and mitochondria from transfected cells. HeLa cells were transfected with a vector expressing untagged vMIA/pUL37x1 and harvested 36 hr after transfection and ER/MAM and mitochondria were isolated. DRMs were then isolated by extraction with 1% Triton X-100 on ice and flotation in discontinuous 5%, 35% and 45% sucrose gradients. Twelve fractions (330μl) were collected from top of the gradient and aliquots (30 μl) of each were subjected to SDS-PAGE and Western analyses with anti-Grp75 (1:1000, Stressgen), Calnexin (1:500; Santa Cruz); HSP60, Mfn2 (1:200; Cell Signal), Prohibitin (1:500, GeneTEX), VDAC (1:1000, Cell Signal), Erlin2 (1:500, a gift from Dr. Stephen Robbins).
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