Subcellular Fractionation Procedures and Metabolic Labeling Using [35S]Sulfate to Isolate Dense Core Secretory Granules from Neuroendocrine Cell Lines
Subcellular fractionation techniques have been developed to allow isolation of a particular subcellular compartments. Typically subcellular fractionation is used as a starting point to characterize the composition of subcellular organelles, but has also been used frequently to provide membranes for cell-free assays that reconstitute a particular intracellular event or process.
Ideally, for both purposes (characterization and cellfree assays) the subcellular compartment should be purified to homogeneity. However, this is difficult to achieve, particularly when the starting material is from cell lines where amounts are limiting. Therefore, it is realistic to aim to achieve a highly enriched fraction (defined here as greater than 90% purity) with a good yield. In addition, it is important to carefully consider the purpose of the fractionation protocol as this will dictate how critical the purity is.
To study the formation of secretory granules in the PC12 cell line, a protocol was developed to identify nascent immature granules by optimizing their separation from the donor compartment, the Golgi complex (Tooze and Huttner, 1990). A combination of velocity-controlled differential and equilibrium density centrifugation achieved a separation of immature secretory granules and the trans-Golgi network (TGN). This protocol was also used to obtain a population highly enriched in immature secretory granules (ISG) and one highly enriched in mature secretory granules (MSG) (Tooze et al., 1991). MSGs are easier to obtain in a more purified form as MSGs are denser than other subcellular compartments. This protocol has been used to characterise the ISG and MSG (Tooze et al., 1991), study low pH-dependent prohormone processing (Urbé et al., 1997), clathrin coat recruitment to ISGs (Dittié et al., 1996), and the role of ADPribosylation factor (ARF) (Austin et al., 2000), sorting of proteins in the ISG (Dittié et al., 1999), cell-free homotypic fusion (Urbé et al., 1998; Wendler et al., 2001), and the recruitment of lipid kinases to ISGs (Panaretou and Tooze, 2002). This protocol has also been used by other researchers to investigate sorting sequences in regulated secretory proteins (Krömer et al., 1996) and with, for example, the AtT20 cell line (Eaton et al., 2000).
II. MATERIALS AND INSTRUMENTATION
PC12 cells are maintained in growth medium consisting of 10% horse serum, 5% fetal calf serum, and Dulbecco's modified Eagle's medium (DMEM) with 3.5 g/liter glucose, penicillin/streptomycin, and 4 mM glutamine (note this is twice the standard concentration) under 10% CO2. Reagents for cell culture are obtained from Sigma Aldrich and GIBCO.
PC12 cells are passaged once a week at a dilution of 1:6. For a standard granule preparation, nine, 245× 245-mm confluent plates of PC12 cells are used. These plates are prepared from five, 175-cm2 flasks harvested with trypsin, seeded, and grown for about 7 days or from nine, 175-cm2 flasks harvested with trypsin, seeded, and grown for about 3 days.
All chemicals are available from Sigma-Aldrich, except sucrose (ultrapure grade Cat. 15503-02), which is from GIBCO.
A cell cracker (EMBL Workshop, EMBL Heidelberg, Germany) with a range of titanium balls. Assemble cell cracker with chosen ball, precool and wash cell cracker with 1 ml of homogenization buffer (HB)/protease inhibitor cocktail (pi) before use. For PC12 cells, use a 18-µm clearance.
Gradients are centrifuged in an SW40 rotor using Beckman ultraclear centrifuge tubes (Cat. No. 344060).
Velocity gradients are prepared using a BioComp gradient master (http://www.biocompinstruments. com), and equilibrium gradients are prepared using a Labconco Auto Densiflow gradient maker (http:// www.labconco.com). Both velocity and equilibrium gradients are collected using the Auto Densiflow.
A cell scraper is made from a silicon rubber bung by cutting the bung with a single-sided razor blade first horizontally (across the widest part) and then vertically in half. The tip of a plastic 10-ml pipette is then inserted into the center of the cut bung.
A. Preparation of a Postnuclear Supernatant
For preparation of sufficient postnuclear supernatant (PNS) to load six SW40 gradients, use nine 245 × 245-mm plates. All solutions must be at 4°C.
B. Velocity Gradient Centrifugation
This method achieves separation of organelles by size rather than density. Thus, larger subcellular compartments will sediment faster during this centrifugation than smaller ones. This is the basis for the separation of ISGs and MSGs from the trans-Golgi network (TGN). In particular, this step is essential for separation of ISGs from the TGN as these organelles have the same equilibrium density.
Sucrose solutions: 0.3M sucrose, 10mM HEPESKOH, pH 7.2, and 1.2M sucrose, 10mM HEPES-KOH, pH 7.2
C. Preparation of ISGs and MSGs by Equilibrium Gradient Centrifugation
This method achieves separation of organelles by density, allowing vesicles of the same size but different densities to be separated. ISG, which have an average diameter of 80nm, can be separated Golgiderived CSVs, which are reported to have a diameter of 50-200nm (Salamero et al., 1990).
Sucrose solutions: 0.8M sucrose, 10 mM HEPESKOH, pH 7.2; 1.0M sucrose, 10 mM HEPES-KOH, pH 7.2; 1.2M sucrose, 10mM HEPES-KOH, pH 7.2; 1.4M sucrose, 10mM HEPES-KOH, pH 7.2; and 1.6M sucrose, 10 mM HEPES-KOH, pH 7.2
D. Other Procedures
The centrifugation procedure can be checked by assaying for TGN, secretory granule, or other compartment-specific markers in each fraction from the velocity and equilibrium gradients. This can be done by Western blotting using antibodies to the marker proteins or by metabolic labelling. For the technique described earlier using PC12 cells, the most accurate method is posttranslational labelling of proteins with [35S]sulfate on tyrosine residues (for a review, see Moore, 2003).
Protein tyrosine sulfation is a posttranslational modification found in some secretory proteins, including secretogranin II (SgII) and an unidentified constitutively secreted heparan sulphated proteoglycan (HSPG). The enzyme tyrosylprotein sulfotransferase responsible for sulfation is a resident TGN protein (Lee and Huttner, 1985). Thus, by incubating or pulsing the cells for a short period of time by the addition of [35S]sulfate, proteins that contain the sulfation motif can be labelled in the TGN and can be used to identify TGN membranes. In addition, exit of sulphated proteins from the TGN into CSVs, which contain HSPG, and ISGs and MSGs, which contain SgII, can be followed accurately during the chase. Identification of CSV and ISGs by sulfate label of the specific markers, followed by subcellular fractionation, can be used to confirm that the cultured cells are correctly sorting regulated and constitutively secreted proteins into separate vesicles (Tooze and Huttner, 1990). Metabolic labelling with sulfate can also be applied more generally (Tooze, 1999). It is advisable to use a scaled down version of the procedure described earlier to avoid having to use large amounts of radioactivity.
To check the ability of PC12 cells to sort regulated proteins using the gradients described earlier, two 150- mm2 dishes of PC12 cells should be prepared for each condition. Ideally the experiment should confirm the position of the TGN on the gradients, identified by a 5-min pulse of sulfate; the CSVs and ISGs, identified by a 5-min pulse followed by a 15-min chase; and the MSGs, identified by a 5-min pulse and 90-min chase. Alternatively, MSGs can be labelled using one-tenth the amount of [35S]sulfate for 6 h and chased overnight.
Austin, C., Hinners, I., and Tooze, S. A. (2000). Direct and GTPdependent interaction of ADP-ribosylation factor 1 with clathrin adaptor protein AP-1 on immature secretory granules. J. Biol. Chem. 275, 21862-21869.
Ditti6, A. S., Hajibagheri, N., and Tooze, S. A. (1996). The AP-1 adaptor complex binds to immature secretory granules from PC12 cells, and is regulated by ADP-ribosylation factor. J. Cell Biol. 132, 523-536.
Ditti6, A. S., Klumperman, J., and Tooze, S. A. (1999). Differential distribution of mannose-6-phosphate receptors and furin in immature secretory granules. J. Cell Sci. 112, 3955-3966.
Eaton, B. A., Haugwitz, M., Lau, D., and Moore, H. P. (2000). Biogenesis of regulated exocytotic carriers in neuroendocrine cells. J. Neurosci. 20, 7334-7344.
Howell, K. E., Schmid, R., Ugelstad, J., and Gruenberg, J. (1989). "Immunoisolation using Magnetic Solid Supports: Subcellular Fractionation for Cell Free Functional Studies," pp. 265-292. Academic Press, New York.
Krömer, A., Samenfeld, P., Loef, I., Huttner, W. B., and Gerdes, H.- H. (1996). Essential role of the granin-loop for sorting to secretory granules is revealed by expression of chromogranin B in the absence of endogenous protein synthesis. J. Cell Biol. 140, 1059-1070.
Lee, R. W. H., and Huttner, W. B. (1985). (Glu62, Ala30, Tyr8)n serves as high-affinity substrate for tyrolsylprotein sulfotransferase; a Golgi enzyme. Proc. Natl. Acad. Sci. USA 82, 6143-6147.
Moore, K. L. (2003). The biology and enzymology of protein tyrosine O-sulfation. J. Biol. Chem. 278, 24243-24246.
Panaretou, C., and Tooze, S. A. (2002). Regulation and recruitment of phosphatidylinositol 4-kinase on immature secretory granules is independent of ADP-ribosylation Factor 1. Biochem. J. 363, 289-295.
Salamero, J., Sztul, E., and Howell, K. (1990). Exocytic transport vesicles generated in vitro from the trans-Golgi network carry secretory and plasma membrane proteins. Proc. Natl. Acad. Sci. USA 87, 7717-7721.
Tooze, S. A. (1999). Metabolic labeling with sulfate. In "Current Protocols in Cell Biology" (J. S. Bonifacino, M. Dasso, J. B. Harford, J. Lippincott-Schwartz, and K. M. Yamada, eds.), Vol. 1, pp. 7.3.1-7.3.7, Wiley, New York.
Tooze, S. A., Flatmark, T., Tooze, J., and Huttner, W. B. (1991). Characterization of the immature secretory granule, an intermediate in granule biogenesis. J. Cell Biol. 115, 1491-1503.
Tooze, S. A., and Huttner, W. B. (1990). Cell-free protein sorting to the regulated and constitutive secretory pathways. Cell 60, 837-847.
Tooze, S. A., and Huttner, W. B. (1992). Cell-free formation of immature secretory granules and constitutive secretory vesicles from trans-Golgi Network. Methods Enzymol. 219, 81-93.
Urbé, S., Ditti6, A., and Tooze, S. A. (1997). pH-dependent processing of secretogranin II by the endopeptidase PC2 in isolated immature secretory granules. Biochem. J. 321, 65-74.
Urbé, S., Page, L. J., and Tooze, S. A. (1998). Homotypic fusion of immature secretory granules during maturation in a cell-free assay. J. Cell Biol. 143, 1831-1844.
Wendler, E, Page, L., Urbé, S., and Tooze, S. A. (2001). Homotypic fusion of immature secretory granules during maturation requires syntaxin 6. Mol. Biol. Cell 12, 1699-1709.
Wu, C. C., MacCoss, M. J., Howell, K. E., and Yates, J. R. III. (2003). A method for the comprehensive proteomic analysis of membrane proteins. Nature Biotechnol. 21, 532-538.
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