This work is supported by the Takasaki University of Health and Welfare.

1. Growing Cells and Addition of Exogenous Antigens
<ol>
	<li>Prepare bOVA using a biotin-protein labeling kit following the manufacturer&#39;s protocol. 
	NOTE: Ordinarily, bOVA contains 2 M biotin per 1 M OVA on average.</li>
	<li>Grow DC2.4 cells in RPMI-1640 supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 100 U/mL penicillin-streptomycin, 55 mM 2-mercaptoethanol, 10 mM HEPES (pH 7.5), and 10% fetal calf serum (hereafter RPMI) at 37 &#176;C in 5% CO2 in a humidified incubator (hereafter without mention, cells are incubated at this condition). It is also possible to use DMEM supplemented with 10% fetal calf serum, 3.7 g/L NaHCO3 (hereafter DMEM) in place of RPMI.</li>
	<li>A day before purification, split the cells into RPMI to 1 x 105 cells/mL in a tissue culture plate. Avoid keeping the cells at a confluent state. DC2.4 cells can highly incorporate exogenous antigens until a semi-confluent state, but rapidly lose the ability after reaching an over-confluent state.</li>
	<li>Before the DC2.4 cells are semi-confluent, add 250 &#181;g/mL of bOVA for 1 x 106 cells/mL and incubate for 2 - 4 h. 
	NOTE: During downstream experiments, all buffers and reagents are kept at 4 &#176;C unless otherwise indicated.</li>
</ol>
2. Preparation of Microsomes
<ol>
	<li>Harvest the DC2.4 cells by gentle pipetting from the tissue plate into a new 50 mL conical tube.</li>
	<li>Centrifuge at 1,000 x g for 5 min, 4 &#176;C and carefully remove the medium with bOVA by aspiration.</li>
	<li>Wash the DC2.4 cells twice with 40 mL of PBS buffer (1.37 mM NaCl, 8.1 mM Na2HPO4, 2.68 mM KCl, 1.47 mM KH2PO4) by centrifugation at 1,000 x g for 5 min, 4 &#176;C and discard the supernatant each time by aspiration.</li>
	<li>Resuspend the pellet in step 2.3 in 1 - 2 mL (1/20-1/10 volume of culture medium) of homogenization medium (0.25 M sucrose, 1 mM EDTA, 10 mM HEPES-NaOH [pH 7.4]) with 1/1,000 volume of protease inhibitor cocktails and transfer the resuspended DC2.4 cells into an ice-cold Dounce homogenizer.</li>
	<li>Disrupt the resuspended DC2.4 cells gently by 10 - 20 strokes with the ice-cold Dounce homogenizer. 
	NOTE: During the disruption step, the Dounce homogenizer is cooled on ice.</li>
	<li>Transfer the cell-disrupted suspension to a new 15 mL conical tube.</li>
	<li>Add 8 - 9 mL homogenization medium to 10 mL total and centrifuge the conical tube for 10 min at 2,000 x g and 4 &#176;C.</li>
	<li>Transfer the supernatant to a new 15-mL conical tube to remove unbroken cells and nuclei, and centrifuge the conical tube again for 10 min at 2,000 x g and 4 &#176;C.</li>
	<li>Transfer the supernatant into a new ultracentrifugation tube and centrifuge for 45 min at 100,000 x g and 4 &#176;C.</li>
	<li>Aspirate the supernatant and resuspend the pellet carefully in 1 - 2 mL of homogenization medium with 1/1,000 volume of protease inhibitor cocktails by pipetting the solution up and down several times to make a microsome fraction for downstream experiments.</li>
	<li>Transfer the microsome fraction into a new 5-mL round bottom tube. 
	NOTE: After this step, it is possible to enrich objective microsomes by iodixanol density gradient centrifugation according to the manufacturer&#39;s protocol, to remove non-specific microsomes. The peak fractions for exogenous antigens are collected and then subjected to the next step of purification (step 3).</li>
</ol>
3. Purification of Microsomes with bOVA Undergoing ERAD
<ol>
	<li>Add 1/100 volume of fresh SA-magnetic beads to the microsome fraction of step 2.11 in the 5 mL round bottom tube. 
	NOTE: Before this step, it is possible to pre-clear the microsome fraction by control-magnetic beads to reduce contaminations of non-specific microsomes.</li>
	<li>Gently mix well and rotate the round bottom tube slowly for 30 min at 4 &#176;C.</li>
	<li>Add 2-3 mL of homogenization medium to 4 mL total into the round bottom tube and gently mix well.</li>
	<li>Place the round bottom tube on a magnetic stand and incubate for 10 min at 4 &#176;C. Since the beads bound to the vesicles are attracted to the magnet, brown micro-beads will gradually accumulate to the tube wall closest to the magnet.</li>
	<li>With the tube remaining in the magnetic stand, carefully discard the supernatants by aspiration to remove the unbound vesicles.</li>
	<li>Wash the magnetic beads bound to the vesicles twice with 5 mL of homogenization medium by the magnetic stand for 10 min at 4 &#176;C and discard the supernatant each time by carefully aspiration. 
	NOTE: Without the magnetic stand, purification by centrifugations at 2,000 x g for 10 min, 4 &#176;C is also available.</li>
	<li>Resuspend the magnetic beads bound to the vesicles carefully in 100 &#181;L homogenization medium by pipetting the solution up and down several times.</li>
	<li>Transfer the resuspended vesicles into a new microtube as the purified microsome for downstream experiments. 
	NOTE: Typically, 50 &#181;L microsome fraction containing 5 - 20 &#181;g proteins from 1 x 107 cells can be isolated.</li>
</ol>
4. Analysis of the Purified Microsomes
<ol>
	<li>Resuspend the magnetic beads bound to the vesicles from step 3.8 by 100 - 200 &#181;L of TNE buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.5 M EDTA, 1% Nonidet P-40) with 1/1,000 volume of protease inhibitor cocktails instead of the homogenization medium.</li>
	<li>Transfer the lysate from step 4.1 to a new 1.5-mL microtube.</li>
	<li>Determine the protein concentration of step 4.2 by using a BCA assay kit per the manufacturer&#39;s protocol. 
	NOTE: Typically, 5 - 10 &#181;L lysate from step 4.2 is enough to determine the protein concentration.</li>
	<li>Transfer the lysate from step 4.2 (2 &#181;g of protein for silver staining and 10 &#181;g of protein for Western blotting) into new microtubes.</li>
	<li>Put the microtubes on a heat block at 95 &#176;C and boil proteins in 1x SDS gel-loading buffer (100 mM Tris-HCl pH 6.8, 200 mM dithiothreitol, 4% SDS, 0.2% bromophenol blue, 20% glycerol) for 5 min.</li>
	<li>Centrifuge the microtubes for 10 min at 21,500 x g and 4 &#176;C. Collect the supernatants into new microtubes to remove the insoluble fractions.</li>
	<li>Analyze the resolved proteins in step 4.6 (2 &#181;g) by standard SD-PAGE.</li>
	<li>Visualize the protein bands by the silver staining using silver staining kit following the manufacturer&#39;s protocol.</li>
	<li>Analyze the resolved proteins in step 4.6 (10 &#181;g) by standard SD-PAGE and Western-blotting. Use the reagent (SA-HRP) per the manufacturer&#39;s protocol and visualize by fluorography.</li>
</ol>
5. In Vitro Reconstitution of ERAD Ubiquitination of bOVA Using Purified Microsomes
<ol>
	<li>Transfer the purified microsomes (5 - 10 &#181;g of protein) from step 3.8 with a 50% volume of reticulocyte lysate (RL), in 1x reaction buffer (50 mM Tris pH 7.4, 3 mM ATP, 0.5 mM MgCl2), and 0.2 pM Flag-tagged ubiquitin in a new microtube. The final volume of this experiment is 20 - 40 &#181;L.</li>
	<li>Incubate the microtube for 2 h at 37 &#176;C. 
	NOTE: If the amount of ubiquitinated bOVA in step 5.12 is too small to detect by Western blotting, add 10 &#181;M of MG132 or 2 &#181;M of lactacystin to inhibit the processing of the ubiquitinated bOVA by proteasomes.</li>
	<li>Stop the reaction by placing the microtube on ice.</li>
	<li>Resolve the microsomes by adding 500 &#181;L TNE buffer with 1/1,000 volume of protease inhibitor cocktails.</li>
	<li>Centrifuge the microtube for 10 min at 21,500 x g and 4 &#176;C. Collect the supernatants into a new microtube to remove the insoluble fractions, which contain ubiquitinated bOVA in DC2.4 before the in vitro ubiquitination assay.</li>
	<li>Transfer the supernatant to a new microtube and add 1/100 volume of new SA-magnetic beads (usually 5 &#181;L for 500 &#181;L of supernatants).</li>
	<li>Gently mix well and rotate the microtube slowly for 30 min at 4 &#176;C.</li>
	<li>Centrifuge the microtube for 10 min at 21,500 x g and 4 &#176;C. Discard the supernatant by aspiration to recover the bOVA and ubiquitinated bOVA bound with the SA-magnetic beads.</li>
	<li>Wash twice the collected SA-magnetic beads with 1 mL of TNE buffer by centrifugation for 10 min at 21,500 x g and 4 &#176;C. Discard the supernatant each time by aspiration.</li>
	<li>Boil the SA-magnetic beads in 1x SDS gel loading buffer for 5 min at 95 &#176;C on a heat-block to resolve the purified proteins by the SA-magnetic beads.</li>
	<li>Centrifuge the microtube for 10 min at 21,500 x g and 4 &#176;C. Collect the supernatants into a new microtube to remove the insoluble fractions.</li>
	<li>Analyze the SA-magnetic beads bound to the proteins by standard SD-PAGE and Western-blotting. The use of antibodies (anti-Flag, anti-multi-Ub, and anti-mouse IgG-HRP) and the reagent (SA-HRP) is per the manufacturer&#39;s protocol and visualized by fluorography.</li>
</ol>

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</ol>

The authors have nothing to disclose.

In previous studies of CP, the incorporated exogenous antigens accumulated in the restricted area of the late endosome or ER by immunofluorescent microscopy16,30,31,32. It is estimated that ERAD-like transport and processing of exogenous antigens are carried out in these specialized areas of the ER or late endosome, as the cellular compartment was identified by sucrose or iodixanol density gradient centrifugation using ubiquitinated bOVA as an ERAD-like processing marker. After stimulating their innate immunity, CP efficiency significantly increased, and the peak fraction for bOVA together with ubiquitinated bOVA migrated to a higher density fraction (our unpublished results). The aims of these experiments were to identify specific molecular markers for the ER or late endosome, which migrated together with bOVA or ubiquitinated bOVA. But in the results, both ER resident molecules and late endosome-resident molecules migrated together with the bOVA and ubiquitinated bOVA, indicating that these experiments were unsuccessful in ascertaining the cellular compartment for ERAD-like transport and processing as the classical ER or late endosome. In these experiments, there were no specific molecules in the endocytotic compartments except bOVA or ubiquitinated bOVA. However, while the exogenous antigen underwent ERAD-like transport, these molecules penetrated the lipid bilayer membrane of ER through translocons, such as Sec61 complex (Figure 1, Figure 2A). The membrane sticking bOVA was selected as a marker of the endocytic compartments, and the microsomes were purified by SA-magnetic beads (Figure 2A, 2B). In these purified microsomes, accumulated bOVA came across ERAD-like ubiquitination and processing under the presence of RL and ATP in vitro (Figure 3A, 3B). Since RL contains all cytosolic molecular apparati, such as ubiquitination-related molecules and proteasomes in addition to molecules for translation and transcription, the addition of RL enables ubiquitination of bOVA in purified microsome. These results indicate that purified microsomes were the objective cellular compartments for ERAD-like transport and processing of exogenous antigens, both containing endosome-specific molecules and ER-resident molecules at the same time; Lamp1 showed both precursor and mature forms in the purified microsome.
This protocol is dependent upon the amount of membrane-penetrating exogenous antigens; it is important to incorporate enough of the exogenous antigens to DC2.4 cells, which shows a high ability to incorporate exogenous antigens in the semi-confluent state. Elongating the 6 - 12 h incubation time for DC2.4 with bOVA increases the amount of incorporated bOVA. The addition of inhibitors for proteasomes, MG132 or lactacystin, moderately increases the amounts of purified microsomes. The preparation of the microsome is one of the most critical steps for this protocol. The free bOVA, which is not incorporated into DC2.4 cells, should be removed by washing the cells with PBS, to prevent non-specific binding of the free bOVA to the microsome fraction. To reduce damages upon membrane compartments, cells are carefully disrupted by the Dounce homogenizer in ice water. Then unbroken cells and nuclei were removed by centrifugation as the microsomes were prepared from the post-nuclear fraction. After preparation of the microsomes, one-step purification by SA-magnetic beads is carried out. In this step, careful washing of the magnet bound microsome is necessary to remove the non-specific binding of other cellular compartments and to not lose the specifically bound microsome.
In this one step purification method, a significant percentage of the target microsome is required. If the ratio of the objective microsome is too small, the amount of purified target microsome decreases by competition with non-specific binding of other cellular compartments. Consequently, under those conditions, the enrichments of the target microsome and the clearance of the non-specific microsome would be required. It is also possible to introduce additional steps before purification by the SA-magnetic beads. One such step is the sucrose or iodixanol density gradient centrifugation of the microsome. Since the prepared microsomes contain all membrane products, it might be possible to enrich the target microsome before SA-magnetic beads purification by selecting the antigen rich fractions for purification. Another possible step is the pre-clearance of the microsome by control-magnetic beads without SA, to reduce the non-specific background associations of microsomes against SA-magnetic beads. In these experiments, both steps improved the purity of the target microsome effectively, but diminished the total amount of purified products, indicating these additional steps are selected as means of further experiments.
It is well known that high-speed centrifugation has been shown to cause the fusion or clotting of intracellular vesicles. These fusions or clottings will produce artificial microsomes, which are derived from the non-specific interaction among different kinds of organelle-derived vesicles. These artificial microsomes statistically contain every membrane molecule such as the mitochondrial resident molecule, Golgi apparatus resident molecule, etc. These purified microsomes did not include the caveosome resident proteins, Golgi apparatus resident proteins, or early endosome-resident proteins, such as caveolin1, GM130, and EEA1, indicating that the SA-purified microsomes are not artificial products derived from the fusion among different kinds of vesicles. Without the high-speed centrifugation, bOVA-sticking vesicles were isolated, but the control experiments from vesicles without bOVA showed higher amounts of non-specific molecules. This indicates that this preliminary method is insufficient for successive experiments and that the high-speed centrifugation step is necessary for this purification protocol.
This protocol applies to other cell types, such as different DC subsets, or other APCs. It is also possible to use the various types of exogenous antigens in different conditions, such as fluorescent proteins, antigen-antibody complex, beads bound antigens, etc. Furthermore, we can purify target microsomes using specific antibodies against the exogenous antigens. Although several additional steps or modifications might be required to apply this protocol to other cells and antigens, it can elucidate the molecular mechanisms of the proteasome-dependent CP by comparing the purified molecules among different experiments. Thus, the described protocol has room for modification and improvement.

The MHC I molecules are expressed on the surface of all nucleated cells, with short antigenic peptides derived from endogenous antigens, which are processed by the ubiquitin-proteasome system in the cytosol1. After processing, antigenic peptides are transported into the endoplasmic reticulum (ER) lumen by the peptide transporter TAP. In the ER lumen, a series of specific chaperones assist the peptide loading and the correct folding of the MHC I complex. This series of molecules is called the peptide-loading complex (PLC), indicating that the ER is a central compartment for peptide loading upon MHC I2. After peptide loading, the MHC I molecules are transported to the cell surface and play a key role in the adaptive immune system as self-markers, and enables the CD8+ cytotoxic T lymphocytes (CTLs) to detect cancer cells or infectious agents by antigenic peptides from non-self proteins3.
In antigen presenting cells (APC), antigenic peptides from exogenous antigens are also presented upon MHC I4,5,6,7,8 via CP, which is mainly carried out by DCs9,10,11. CP is essential both for the activation of na&#239;ve CD8+ T cells and memory CD8+ T cells into anti-infectious and anti-tumoral CTLs12,13, and in the maintenance of immune tolerance by the inactivating of self-acting na&#239;ve T cells14,15. The CP plays many important roles in the adaptive immune system, however the molecular mechanisms of CP have yet to be described in detail. Previous studies of CP revealed that exogenous antigens were localized both in the ER and the endosome and were processed by ERAD, suggesting that exogenous antigens are transported from the endosome to the ER for ERAD-like processing and peptide loading16. However, accumulating evidence indicates that the peptide loading of CP is carried out not in the ER but rather in non-classical endocytic compartments, which also have distinctive features of the ER (Figure 1)17,18,19,20,21. To avoid degradation of the antigenic peptide precursors by the high activity of aminopeptidase22 in the cytosol, processing and peptide loading in CP occurs in the proximal area of these non-classical endocytic compartments (Figure 1). Though the characterizations of these endocytic compartments are controversial, there are no existing specific molecules other than exogenous antigens in this compartment.
ERAD is a cellular pathway, which specifically removes misfolded proteins from the ER. In the ERAD pathway, misfolded proteins are retrogradely transported through the ER membrane to the cytoplasm and processed by the ubiquitin-proteasome system23,24,25. When large molecules, such as proteins, are transported through the lipid bilayer, these molecules pass through a molecular apparatus called a translocon, such as the Sec61 complex and Derlin complex in the ER26, and the Tom complex and Tim complex in the mitochondria27. When exogenously-added antigens are transported through the ER membrane, they must penetrate the lipid bilayer in complex with translocons, such as the Sec61 complex. The method described here purified the targeted vesicle by utilizing these membrane-penetrating molecules as markers for the endocytic compartments.
The method described here is a new vesicle purification protocol using the DC-like cell line DC2.428 and biotinylated ovalbumin (bOVA) as an exogenous antigen. The endocytic compartments were purified by streptavidin (SA)-magnetic beads using the membrane-penetrating bOVA as a maker. In this purified microsome, some exogenously added bOVA was still preserved in membrane fractions but were transported to the outside of microsome, and then ubiquitinated and processed in vitro29. This purified microsome contained not only endocytic compartment-specific proteins but also ER-resident proteins for ERAD and the peptide loading complex; suggesting that the cellular compartment is the prospective endocytic compartment for CP29. This protocol is not dependent on the kind of exogenous antigens, and is also applicable for other DC subsets and other cell types, such as macrophages, B cells, and endothelial cells, to clarify the precise molecular mechanism of DCs for proficient CP.

<table><tbody><tr><td>RPMI 1640</td><td>gibco by life technologies</td><td>11875-093</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Equitech bio</td><td>SFB30</td><td></td></tr><tr><td>Sodium pyruvate</td><td>gibco by life technologies</td><td>11360-070</td><td></td></tr><tr><td>MEM non-essential amino acids</td><td>gibco by life technologies</td><td>11140-050</td><td></td></tr><tr><td>HEPES</td><td>gibco by life technologies</td><td>15630-080</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>gibco by life technologies</td><td>21985-023</td><td></td></tr><tr><td>L-glutamine</td><td>gibco by life technologies</td><td>25030-164</td><td></td></tr><tr><td>Penisicillin-Sreptomycin</td><td>gibco by life technologies</td><td>15140-122</td><td></td></tr><tr><td>DMEM</td><td>gibco by life technologies</td><td>12100-46</td><td></td></tr><tr><td>OVA</td><td>SIGMA</td><td>A5503</td><td></td></tr><tr><td>Biotin-protein labelling kit</td><td>Thermo Fisher Scientific</td><td>F6347</td><td></td></tr><tr><td>MG-132</td><td>Santa Cruz Biotechnology</td><td>201270</td><td></td></tr><tr><td>lactacystin</td><td>SIGMA</td><td>L6785</td><td></td></tr><tr><td>Dounce homogenizer</td><td>IUCHI</td><td>131703</td><td></td></tr><tr><td>protease inhibitor cocktails</td><td>SIGMA</td><td>P8340</td><td></td></tr><tr><td>iodixanol</td><td>Cosmo bio</td><td>1114542</td><td></td></tr><tr><td>SA-magnetic beads</td><td>New England Biolabs</td><td>201270</td><td></td></tr><tr><td>control magnetic beads</td><td>Chemagen</td><td>M-PVA012</td><td></td></tr><tr><td>magnetic stand</td><td>BD Biosciences</td><td>552311</td><td></td></tr><tr><td>BCA protein assay kit</td><td>Thermo Fisher Scientific</td><td>23225</td><td></td></tr><tr><td>silver staining kits</td><td>Cosmo bio</td><td>423413</td><td></td></tr><tr><td>Reticulocyte Lysate</td><td>Promega</td><td>1730714</td><td></td></tr><tr><td>Flag-tagged ubiquitin</td><td>SIGMA</td><td>U5382</td><td></td></tr><tr><td>anti-ovalbumin (OVA,mouse)</td><td>Antibody Shop</td><td>HYB 094-06</td><td></td></tr><tr><td>ant-multi-ubiquitin (mouse)</td><td>MBL</td><td>D058&amp;minus;3</td><td></td></tr><tr><td>anti-Flag (mouse)</td><td>SIGMA</td><td>F3165</td><td></td></tr><tr><td>trypsin</td><td>SIGMA</td><td>85450C</td><td></td></tr></tbody></table>

purification of the membrane compartment for endoplasmic reticulum-associated degradation of exogenous antigens in cross-presentation

Dendritic cells (DCs) are highly capable of processing and presenting internalized exogenous antigens upon major histocompatibility class (MHC) I molecules also known as cross-presentation (CP). CP plays an important role not only in the stimulation of na&#239;ve CD8+ T cells and memory CD8+ T cells for infectious and tumor immunity but also in the inactivation of self-acting na&#239;ve T cells by T cell anergy or T cell deletion. Although the critical molecular mechanism of CP remains to be elucidated, accumulating evidence indicates that exogenous antigens are processed through endoplasmic reticulum-associated degradation (ERAD) after export from non-classical endocytic compartments. Until recently, characterizations of these endocytic compartments were limited because there were no specific molecular markers other than exogenous antigens. The method described here is a new vesicle isolation protocol, which allows for the purification of these endocytic compartments. Using this purified microsome, we reconstituted the ERAD-like transport, ubiquitination, and processing of the exogenous antigen in vitro, suggesting that the ubiquitin-proteasome system processed the exogenous antigen after export from this cellular compartment. This protocol can be further applied to other cell types to clarify the molecular mechanism of CP.

To elucidate the molecular mechanism of CP, it is necessary to identify the cellular compartments, where exogenous antigens undergo ERAD-like transport and processing. While observations by immunofluorescent microscopy or by electron microscopy identified the cellular compartment where exogenous antigens accumulated16,17,18,19,30,31,32,33, the cellular compartments for ERAD-like processing of exogenous antigens are not clearly defined. Recently it was shown that non-classical endosomes with ER resident molecules were responsible for CP34, but these cellular compartments were unpurified. The difficulty in isolating and purifying the cellular compartments can be attributed to the fact that exogenous antigens are localized both in the endosome and ER-like compartments and that there is no identifying molecule for these endocytic compartments other than exogenous antigens. However, the condition of exogenous antigens undergoing ERAD-like transport is different from the steady state; in the transport across lipid bimolecular membrane, exogenous antigens penetrate the membrane via translocons, such as Sec61 (Figure 2A). Thus, when using bOVA as an exogenous antigen, bOVA should be associated with Sec61. Since membrane-associated bOVA specifically bound with SA, the microsome prepared from DC2.4, which was pretreated with bOVA, could be isolated distinctively by SA-magnetic beads (Figure 2A). Then equivalent amounts of proteins from purified microsomes with or without pre-incubation of bOVA were resolved by SDS-PAGE followed by silver staining and Western blotting with SA-HRP (Figure 2B, 2C). As shown in Figure 2B and Figure 2C, the isolated microsomes contained several unique proteins that were purified dependent upon exogenously added bOVA and SA-magnetic beads. In addition to these unique proteins, isolated microsomes also contained nonspecific proteins, which bound to SA-magnetic beads with or without bOVA. Treatment of the microsome with trypsin before purification by the magnet prevented the purification of microsomes (Figure 2D), indicating that the purification methods depended on the presence of membrane-penetrating bOVA.
The purified microsomes showed the ability to ubiquitinate the incorporated bOVA in vitro, under the presence of RLs (Figure 3A). The amounts of bOVA and poly-ubiquitinated bOVA were augmented in the presence of MG132 (Figure 3B), indicating that the incorporated bOVA was processed by the ERAD system and that our purified microsomes contained ERAD machinery proteins.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55949/55949fig1.jpg" /> 
Figure 1: Intracellular Pathways for CP in DCs. In DCs, exogenous antigens are transported into non-classical endocytic compartments, which also contain ER-resident molecules in addition to molecules of the classical late endosome. In this compartment, exogenous antigens are exported into cytosol through translocons such as Sec61. In the cytosol, exogenous antigens are processed by the ubiquitin-proteasome system into antigenic peptides as ERAD substrates. Antigenic peptides are transported into same or adjacent non-classical endocytic compartments, or adjacent ER through TAP transporter and then loaded on the MHC I molecules by PLC. <a href="http://cloudflare.jove.com/files/ftp_upload/55949/55949fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55949/55949fig2.jpg" /> 
Figure 2: Purification of Microsomes with bOVA Undergoing ERAD. (A) A schematic model of purification of microsomes with bOVA undergoing ERAD. bOVA is associated with the membrane through the Sec61 translocon and targeted with SA-magnetic beads. (B) Microsomes with (+) or without (-) prior addition of bOVA were purified with (+) or without (-) SA-magnetic beads. Proteins (2 &#181;g) or corresponding volumes of purified proteins were resolved on 7.5 - 15% SDS-PAGE, and silver staining was used to visualize protein bands. Triangles on the right side indicate nonspecific proteins binding to the SA-magnetic beads. Triangles with asterisks indicate unique proteins found only in the presence of exogenously added bOVA and SA-magnetic beads. The arrow shows bOVA. (C) Microsomes with (+) or without (-) prior addition of bOVA were purified with (+) or without (-) SA-magnetic beads. Proteins (10 &#181;g) or corresponding volumes of purified proteins were resolved on 7.5 - 15% SDS-PAGE, and subjected to Western blotting with SA-HRP. P.N.: post-nuclear fraction. Asterisks in the right indicate non-specific bands with SA-HRP. Equivalent results were attained by at least three independent assays. (D) Microsomes with prior addition of bOVA were purified with SA-magnetic beads. Microsomes were treated with (+) or without (-) trypsin and TX-100 before purification (left two lanes) or after purification (right two lanes). Proteins (2 &#181;g) or corresponding volumes of purified proteins were resolved on 7.5 - 15% SDS-PAGE, and silver staining was used to visualize protein bands. Triangles on the right side indicate nonspecific proteins binding to the SA-magnetic beads. Triangles with asterisks indicate unique proteins found only in the presence of exogenously added bOVA and SA-magnetic beads. Equivalent results were attained by at least three independent assays. Reprinted with permission from reference28. <a href="http://cloudflare.jove.com/files/ftp_upload/55949/55949fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55949/55949fig3.jpg" /> 
Figure 3: In Vitro Reconstitution of Processing and Ubiquitination using OVA in Purified Microsomes. (A) Purified microsomes with (+) or without (-) prior addition of bOVA were treated with (+) or without (-) RL and Flag-Ub for 1 h and were solubilized using TNE. bOVA was purified with SA-magnetic beads and subjected to Western blotting with the indicated antibodies. Asterisks in the right indicate non-specific bands with SA-HRP. Equivalent results were attained by at least three independent assays. (B) Purified microsomes were treated with (+) or without (-) RL, Flag-Ub, and MG132 for 1 h and were then solubilized using TNE. bOVA was purified with SA-magnetic beads and subjected to Western blotting with SA-Flag. Asterisks in the right indicate non-specific bands with SA-HRP. Equivalent results were attained by at least three independent assays. Reprinted with permission from reference28. <a href="http://cloudflare.jove.com/files/ftp_upload/55949/55949fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation at JoVE.com

Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation

The method described here is a new vesicle isolation protocol, which allows for the purification of the cellular compartments where exogenous antigens are processed by endoplasmic reticulum-associated degradation in cross-presentation.

Dendritic cells (DCs) are highly capable of processing and presenting internalized exogenous antigens upon major histocompatibility class (MHC) I ...

purification-membrane-compartment-for-endoplasmic-reticulum

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

8.2K Views.  Takasaki University of Health and Welfare. The method described here is a new vesicle isolation protocol, which allows for the purification of the cellular compartments where exogenous antigens are processed by endoplasmic reticulum-associated degradation in cross-presentation.

Video: Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation

Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a large extracellular domain and a short cytosolic domain termed the APP intracellular domain (AICD). During maturation through the secretory pathway, APP can be cleaved by proteases termed &alpha;, &beta;, and &gamma;-secretases1. Sequential proteolytic cleavage of APP with &beta; and &gamma;-secretases leads to the production of a small proteolytic peptide, termed A&beta;, which is amyloidogenic and the core constituent of senile plaques. The AICD is also liberated from the membrane after secretase processing, and through interactions with Fe65 and Tip60, can translocate to the nucleus to participate in transcription regulation of multiple target genes2,3. Protein-protein interactions involving the AICD may affect trafficking, processing, and cellular functions of holo-APP and its C-terminal fragments. We have recently shown that AICD can aggregate in vitro, and this process is inhibited by the AD-implicated molecular chaperone ubiquilin-14. Consistent with these findings, the AICD has exposed hydrophobic domains and is intrinsically disordered in vitro5,6, however it obtains stable secondary structure when bound to Fe657. We have proposed that ubiquilin-1 prevents inappropriate inter- and intramolecular interactions of AICD, preventing aggregation in vitro and in intact cells4. While most studies focus on the role of APP in the pathogenesis of AD, the role of AICD in this process is not clear. Expression of AICD has been shown to induce apoptosis8, to modulate signaling pathways9, and to regulate calcium signaling10. Over-expression of AICD and Fe65 in a transgenic mouse model induces Alzheimer's like pathology11, and recently AICD has been detected in brain lysates by western blotting when using appropriate antigen retrieval techniques12. To facilitate structural, biochemical, and biophysical studies of the AICD, we have developed a procedure to produce recombinantly large amounts of highly pure AICD protein. We further describe a method for inducing the in vitro thermal aggregation of AICD and analysis by atomic force microscopy. The methods described are useful for biochemical, biophysical, and structural characterization of the AICD and the effects of molecular chaperones on AICD aggregation.

A method for large-scale purification of the APP intracellular domain (AICD) is described. We also describe methodology to induce in vitro AICD aggregation and visualization by atomic force microscopy. The methods described are useful for biochemical/structural characterization of the AICD and the effects of molecular chaperones on its aggregation.

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a ...

Medicine

Mitochondria-associated ER Membranes (MAMs) and Glycosphingolipid Enriched Microdomains (GEMs): Isolation from Mouse Brain

Intracellular organelles are highly dynamic structures with varying shape and composition, which are subjected to cell-specific intrinsic and extrinsic cues. Their membranes are often juxtaposed at defined contact sites, which become hubs for the exchange of signaling molecules and membrane components1,2,3,4. The inter-organellar membrane microdomains that are formed between the endoplasmic reticulum (ER) and the mitochondria at the opening of the IP3-sensitive Ca2+ channel are known as the mitochondria associated-ER membranes or MAMs4,5,6. The protein/lipid composition and biochemical properties of these membrane contact sites have been extensively studied particularly in relation to their role in regulating intracellular Ca2+ 4,5,6. The ER serves as the primary store of intracellular Ca2+, and in this capacity regulates a myriad of cellular processes downstream of Ca2+ signaling, including post-translational protein folding and protein maturation7. Mitochondria, on the other hand, maintain Ca2+ homeostasis, by buffering cytosolic Ca2+ concentration thereby preventing the initiation of apoptotic pathways downstream of Ca2+ unbalance4,8. The dynamic nature of the MAMs makes them ideal sites to dissect basic cellular mechanisms, including Ca2+ signaling and regulation of mitochondrial Ca2+ concentration, lipid biosynthesis and transport, energy metabolism and cell survival 4,9,10,11,12. Several protocols have been described for the purification of these microdomains from liver tissue and cultured cells13,14. 
	Taking previously published methods into account, we have adapted a protocol for the isolation of mitochondria and MAMs from the adult mouse brain. To this procedure we have added an extra purification step, namely a Triton X100 extraction, which enables the isolation of the glycosphingolipid enriched microdomain (GEM) fraction of the MAMs. These GEM preparations share several protein components with caveolae and lipid rafts, derived from the plasma membrane or other intracellular membranes, and are proposed to function as gathering points for the clustering of receptor proteins and for protein&#x2013;protein interactions4,15.

Mitochondria-associated ER Membranes MAMs and Glycosphingolipid Enriched Microdomains GEMs: Isolation from Mouse Brain

This procedure illustrates how to isolate from the adult mouse brain the mitochondria-associated ER membranes or MAMs and the glycosphingolipid-enriched microdomain fractions from MAMs and mitochondrial preparations.

Intracellular organelles are highly dynamic structures with varying shape and composition, which are subjected to cell-specific intrinsic and extrinsic ...

Neuroscience

Reconstitution of Msp1 Extraction Activity with Fully Purified Components

As the center for oxidative phosphorylation and apoptotic regulation, mitochondria play a vital role in human health. Proper mitochondrial function depends on a robust quality control system to maintain protein homeostasis (proteostasis). Declines in mitochondrial proteostasis have been linked to cancer, aging, neurodegeneration, and many other diseases. Msp1 is a AAA+ ATPase anchored in the outer mitochondrial membrane that maintains proteostasis by removing mislocalized tail-anchored proteins. Using purified components reconstituted into proteoliposomes, we have shown that Msp1 is necessary and sufficient to extract a model tail-anchored protein from a lipid bilayer. Our simplified reconstituted system overcomes several of the technical barriers that have hindered detailed study of membrane protein extraction. Here, we provide detailed methods for the generation of liposomes, membrane protein reconstitution, and the Msp1 extraction assay.

Here, we present a detailed protocol for reconstitution of Msp1 extraction activity with fully purified components in defined proteoliposomes.

As the center for oxidative phosphorylation and apoptotic regulation, mitochondria play a vital role in human health. Proper mitochondrial function ...

Biochemistry

Spontaneous Formation and Rearrangement of Artificial Lipid Nanotube Networks as a Bottom-Up Model for Endoplasmic Reticulum

We present a convenient method to form a bottom-up structural organelle model for the endoplasmic reticulum (ER). The model consists of highly dense lipidic nanotubes that are, in terms of morphology and dynamics, reminiscent of ER. The networks are derived from phospholipid double bilayer membrane patches adhering to a transparent Al2O3 substrate. The adhesion is mediated by Ca2+ in the ambient buffer. Subsequent depletion of Ca2+ by means of BAPTA/EDTA causes retraction of the membrane, resulting in spontaneous lipid nanotube network formation. The method only comprises phospholipids and microfabricated surfaces for simple formation of an ER model and does not require the addition of proteins or chemical energy (e.g., GTP or ATP). In contrast to the 3D morphology of the cellular endoplasmic reticulum, the model is two-dimensional (albeit the nanotube dimensions, geometry, structure, and dynamics are maintained). This unique in vitro ER model consists of only a few components, is easy to construct, and can be observed under a light microscope. The resulting structure can be further decorated for additional functionality, such as the addition of ER-associated proteins or particles to study transport phenomena among the tubes. The artificial networks described here are suitable structural models for the cellular ER, whose unique characteristic morphology has been shown to be related to its biological function, whereas details regarding formation of the tubular domain and rearrangements within are still not completely understood. We note that this method uses Al2O3 thin-film-coated microscopy coverslips, which are commercially available but require special orders. Therefore, it is advisable to have access to a microfabrication facility for preparation.

Solid-supported, protein-free, double phospholipid bilayer membranes (DLBM) can be transformed into complex and dynamic lipid nanotube networks and can serve as 2D bottom-up models of the endoplasmic reticulum.

We present a convenient method to form a bottom-up structural organelle model for the endoplasmic reticulum (ER). The model consists of highly dense ...

Bioengineering

Purification of the Sarco-Endoplasmic Reticulum Ca2+-ATPase from Rabbit Muscle

Some P-type ATPases, such as sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), are inherently labile membrane proteins that require specific physicochemical conditions during purification to obtain them with high purity and structural quality and in a catalytically active form. The disaccharide trehalose is a compatible solute that is synthesized and accumulated in high concentrations in the yeast cytoplasm to stabilize the membranes and proteins. The use of trehalose as an additive in the protocol for the purification of plasma membrane H+-ATPase results in a high-quality preparation, the hexameric structure of which is shown by biochemical analytical methods. Trehalose can, therefore, be used as a stabilizing additive for the purification of membrane proteins (P-ATPases). This protocol describes the modification of the classical protocol for SERCA purification by subjecting SERCA to centrifugation on a trehalose concentration gradient. The inclusion of this carbohydrate led to the purification of SERCA in a catalytically active form with high purity and, importantly, in a stable form. Partial biochemical characterization of the purified SERCA (SDS-PAGE, enzyme kinetics, FITC labeling, circular dichroism spectroscopy) showed that the enzyme is suitable for functional and structural studies. The use of trehalose in the purification protocol of P-type ATPases and other labile membrane (and cytosolic) proteins is suggested.

This protocol describes an improved SERCA purification method, which includes the disaccharide trehalose in the final centrifugation step. This carbohydrate stabilizes proteins under harsh conditions. The purified SERCA was catalytically active and displayed high purity, making it suitable for structural and functional studies.

Some P-type ATPases, such as sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), are inherently labile membrane proteins that require specific ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

Biology

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Regulation of protein abundance is crucial to virtually every cellular process. Protein abundance reflects the integration of the rates of protein synthesis and protein degradation. Many assays reporting on protein abundance (e.g., single-time point western blotting, flow cytometry, fluorescence microscopy, or growth-based reporter assays) do not allow discrimination of the relative effects of translation and proteolysis on protein levels. This article describes the use of cycloheximide chase followed by western blotting to specifically analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast). In this procedure, yeast cells are incubated in the presence of the translational inhibitor cycloheximide. Aliquots of cells are collected immediately after and at specific time points following addition of cycloheximide. Cells are lysed, and the lysates are separated by polyacrylamide gel electrophoresis for western blot analysis of protein abundance at each time point. The cycloheximide chase procedure permits visualization of the degradation kinetics of the steady state population of a variety of cellular proteins. The procedure may be used to investigate the genetic requirements for and environmental influences on protein degradation.

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Protein abundance reflects the rates of both protein synthesis and protein degradation. This article describes the use of cycloheximide chase followed by western blotting to analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast).

Regulation of protein abundance is crucial to virtually every cellular process. Protein abundance reflects the integration of the rates of protein ...

The Endoplasmic Reticulum

The endoplasmic reticulum or ER makes up for more than half of the membranes in a cell and accounts for 10% of total cell volume. It is also the primary protein and lipid synthesis factory for most cell organelles, such as the Golgi apparatus, lysosomes, secretory vesicles, and the plasma membrane. Despite being the most extensive and functionally complex subcellular organelle, ER was the last to be discovered. After years of deliberation, Keith Porter and George Palade in the year 1954, produced the first high-resolution electron microscope images to affirm the presence of ER in the eukaryotic cell.
The ER membranes were identified as sites for protein synthesis and crucial for intracellular transport using radio-labeled and fluorescent-labeled amino acids. The isolation of ER is a tricky task as it forms an intricate mesh with other subcellular organelles. However, on cell homogenization, the disrupted ER membranes reseal into small closed vesicles called microsomes. These vesicles form a microsystem capable of sustaining all ER-related functions like protein and lipid synthesis, calcium signaling, and glycosylation. Subcellular fractionation is the best and often-used technique for the purification of these membranes. When separating using a sucrose gradient, the rough ER microsomes sediments at a higher density than smooth ER microsomes.
The ER network in a cell is dynamic. It is constantly shape-shifting along with the cytoskeleton to bolster mechanical support for the cell structure. Despite the distinct cisternae and tubule morphologies of the ER membrane, interconversion between the two is possible and is governed by the expression of membrane proteins. The ER network rearranges by tubule growth, retraction, and fusion of adjacent ER-ER membranes.
As mentioned above, the ER network is required for optimal overall cellular health. Disruption of ER morphology is linked to pathological conditions, including neurological disorders like Alzheimer&#39;s disease, hereditary spastic paraplegia, and viral infections like hepatitis C virus, and dengue virus.

The endoplasmic reticulum or ER makes up for more than half of the membranes in a cell and accounts for 10% of total cell volume. It is also the primary ...

Education

JoVE Core

Cell Biology

Unit 1

Transmembrane Transport in Endoplasmic Reticulum and Peroxisomes

KEY TERMS AND CONCEPTS

ER Retrieval Pathway

In the secretory pathway, vesicles transport proteins from one cellular compartment to another in forward transport to deliver the protein to its correct location. Occasionally, misfolded proteins and incorrect proteins escape their original compartments, and a retrieval pathway is used to return the escaped proteins to their original compartment.
The ER uses many checkpoints to prevent the entry of incorrectly folded or a resident protein as cargo onto a transport vesicle. These mechanisms include cargo selectivity of the receptors, aggregation of functionally similar proteins, and retention sequences on resident proteins.
Biochemical studies characterizing the cargo-sorting receptors have shown that they are ligand-specific and bind only at specific pH (pH 5.0 in yeast and mammals). Functionally similar ER-resident can proteins aggregate to form large complexes that cannot be loaded onto transport vesicles, thus avoiding the accidental escape of these proteins from the organelle. Additionally, the membrane-spanning domain of several ER and Golgi proteins contain a retention sequence that marks these proteins for staying back in the ER, so receptors do not bind them.
If an ER-resident protein escapes these checkpoints and gets loaded onto a Golgi-bound vesicle, a specific amino acid sequence called a retrieval signal helps the protein get recognized and transported back to the ER. The receptors involved in retrieval mechanisms recognize the retrieval signals at the carboxyl terminus of soluble proteins of the ER and in the cytoplasmic domain of some ER and Golgi membrane proteins. The receptors selectively capture such proteins and package them in vesicles that transport them back to the ER.
Two well-characterized retrieval signals are the carboxy-terminal tetrapeptide KDEL and KKXX found in many ER lumen resident proteins. BiP, a molecular chaperone, plays a vital role in preventing the aggregation of misfolded proteins. Interaction between BiP and unfolded proteins is mediated by a substrate-binding domain and a nucleotide-binding domain for ATPase activity. KDEL receptor in the post-ER compartments such as ERGIC and Golgi binds with the carboxyl-terminal retrieval signal of BiP and returns BiP to the ER via COPI vesicles.

In the secretory pathway, vesicles transport proteins from one cellular compartment to another in forward transport to deliver the protein to its correct ...

Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation

Summary

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Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

The Endoplasmic Reticulum

ER Retrieval Pathway