RESEARCH ARTICLE 1397 Cell-specific targeting of caveolin-1 to caveolae, secretory vesicles, cytoplasm or mitochondria Wei-Ping Li, Pingsheng Liu, Brian K. Pilcher and Richard G. W. Anderson* Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9039, USA *Author for correspondence (e-mail: [email protected]) Accepted 15 January 2001 Journal of Cell Science 114, 1397-1408 © The Company of Biologists Ltd SUMMARY In commonly used tissue culture cells, caveolin-1 is accumulate caveolin-1 in modified mitochondria. The embedded in caveolae membranes. It appears to reach this cytosolic and the secreted forms appear to be incorporated location after being cotranslationally inserted into ER into a soluble, lipid complex. We conclude that caveolin-1 membranes, processed in the Golgi and shipped to the cell can be targeted to a variety of intracellular destinations, surface. We now report that caveolae are not the preferred which suggests a novel mechanism for the intracellular location for caveolin-1 in all cell types. Skeletal muscle cells traffic of this protein. and keratinocytes target caveolin-1 to the cytosol while in exocrine and endocrine cells it accumulates in the secretory pathway. We also found that airway epithelial cells Key words: Caveolin-1, Intracellular trafficking, Caveolae INTRODUCTION will target the molecule to caveolae by determining with immunocytochemistry if caveolin-1 in different tissue cells is Caveolin-1 was originally identified as a novel tyrosine kinase always in this membrane domain. Unexpectedly, we found substrate in Rous sarcoma transformed cells (Glenney, 1989). examples of cells that preferentially target caveolin-1 to either Immunogold cytochemistry localized the protein to the the cytoplasm, mitochondria or elements of the secretory distinctive striated coat structure that decorates the inner pathway but had little caveolin-1 in invaginated caveolae. The membrane surface of fibroblast caveolae (Rothberg et al., 1992) behavior of caveolin-1 in these cells suggests the existence of and to elements of the Golgi apparatus (Kurzchalia et al., 1992). a novel pathway of intracellular and extracellular molecular Studies carried out concurrently implicated caveolin-1 in the trafficking that may be specialized for delivering lipids to sorting of molecules during vesicular trafficking to the apical multiple cellular compartments. surface of polarized epithelial cells (Kurzchalia et al., 1992). Subsequently, several laboratories used caveolin-1 as an integral protein marker to prepare cell fractions enriched in caveolae MATERIALS AND METHODS (reviewed in Anderson, 1998). Several methods of purification yield vesicles that are approximately the size of caveolae (Smart Cyclophilin A pAb was purchased from ABR (Golden, CO, USA). et al., 1995; Westermann et al., 1999), sometimes with an ApoA 1 pAb was a gift from Dr Helen Hobbs (University of Texas apparent striated coat (Chang et al., 1994). These and other Southwestern Medical Center, USA). Purified human HDL was studies suggest that caveolin-1 plays a role in the traffic of prepared by standard methods. OptiPrep was purchased from caveolae and caveolae-related membranes within the cell Accurate Chemical & Scientific Corporation (Westbury, NY, USA). (Anderson, 1993; Anderson, 1998). Caveolin-2 and caveolin-3 Caveolin-1 pAb and mAb were either raised in our laboratory or obtained from Transduction Laboratories (Lexington, KY, USA). are two homologous proteins that may have similar functions Caveolin-2 mAb and caveolin-3 pAb were obtained from to caveolin-1 (Smart et al., 1999). Transduction Laboratories (Lexington, KY, USA). FITC-goat anti- Studies on the function of caveolin-1 follow two main rabbit IgG was from Zymed Laboratories (San Francisco, CA, USA). themes. One idea is that caveolin-1 is a scaffolding protein Protein A gold was from Dr J. W. Slot (Utrecht University, The involved in organizing the activity of multiple signaling Netherlands). BSA, proline, leupeptin, soybean trypsin inhibitor, molecules in caveolae (Okamoto et al., 1998). The other is pepstatin A, Dulbecco’s modified Eagle’s Medium (DMEM), based on the cholesterol- (Murata et al., 1995) and fatty acid- benzamidine, adenine-free base, human apo-transferrin, aprotinin, L- (Trigatti et al., 1999) binding properties of caveolin-1 and the ascorbic acid, bovine pancreatic insulin, ethylenediaminetetraacetic possibility that it mediates the intracellular transport of lipids acid (EDTA), ethyleneglycoltetraacetic acid (EGTA), hydrocortisone, such as cholesterol (Smart et al., 1996). The exact function of ATP, secretin, dexamethasone, cholecystokinin, mitomycin C, sodium chloride, sodium deoxycholate, sodium fluoride, sodium vanadate and a protein is often reflected in how the molecule is used 3,3′,5-triiodo-L-thyronine sodium salt were from Sigma (St Louis, by various tissue cells. If, for example, caveolin-1 were a MO, USA). A23187 was from Biomol (Plymouth Meeting, PA, USA). scaffolding protein, then the molecule should be in caveolae- Bio-Rad Protein Assay was from Bio-Rad Laboratories (Hercules, related structures whenever it is expressed. In the current study, CA, USA). F-12K medium was from GibcoBRL (Grand Island, NY, we tested the hypothesis that all cells expressing caveolin-1 USA). Fetal bovine serum (FBS) and fetal calf serum (FCS) were 1398 JOURNAL OF CELL SCIENCE 114 (7) from HyClone (Logan, UT, USA). C57Bl/6J mice were from Jackson fibroblast-populated collagen gels (2×104 cells/ml, rat tail type I Laboratory (Bar Harbor, MI, USA). Sprague Dawley rats were from collagen) and placed into Transwell™ polycarbonate cell culture Harlan Sprague Dawley (Indianapolis, IN, USA). Bovine type I inserts (4.0 µm pore size). Following keratinocyte attachment (approx. collagen (Vitrogen-100) was purchased from Celltrix Laboratories 4 hours), the cloning cylinders were removed and cultures were (Palo Alto, CA, USA) and rat tail type I collagen was obtained from immersed in FAD medium containing 50 µg/ml L-ascorbic acid. Upstate Biotechnology (Lake Placid, NY, USA). DMEM/F-12 Following a 6 day culture period to allow the keratinocytes to reach medium, recombinant human epidermal growth factor, and confluence, the cultured skin equivalents were raised to the air-liquid penicillin/streptomycin were obtained from Life Technologies, Inc. interface to stimulate keratinocyte differentiation and cornification. (Rockville, MD, USA). Recombinant Cholera toxin B subunit was FAD medium with additives was replaced every 2 days until cultured obtained from Calbiochem-Novabiochem corporation (Cambridge, skin equivalents were harvested. MA, USA). Borosilicate cloning cylinders (8 mm inner diameter) were purchased form BellCo Glass, Inc. (Vineland, NJ, USA). Isolation of caveolae Transwell™ polycarbonate cell culture inserts (4.0 µm pore size) were Caveolae were isolated by the method of Smart et al. (Smart et al., obtained from Corning Costar Corp. (Cambridge, MA, USA). Hybond 1995). Briefly, confluent normal human fibroblasts were collected in N+ blotting membrane, Rediprime II random priming label and ECL hypotonic buffer and dounced 20 times on ice. Plasma membrane (PM) immunoblotting systems were purchased from Amersham Pharmacia was isolated on a 30% Percoll gradient from the post-nuclear Biotech, Inc. (Piscataway, NJ, USA). [γ32P]-dCTP was obtained from supernatant (PNS) and then sonicated. The sonicated sample was NEN Life Science Products, Inc. (Boston, MA, USA). Immobilon-P mixed with OptiPrep (final OptiPrep concentration, 23%) in a TH641 polyvinylidene difluoride (PVDF) membrane was purchased from tube. A linear 20%-10% OptiPrep gradient was overlaid on the sample Millipore Corp. (Bedford, MA, USA). and centrifuged at 52,000 g for 90 minutes at 4°C. The bottom 1 ml Buffer A: 20 mM Tricine, pH 7.8, 250 mM sucrose, 1 mM EDTA. was designated non-caveolae membranes (NCM). The top 5 ml were Buffer B: 20 mM Tris-HCl, pH 7.6, 130 mM NaCl, 0.2% Tween- collected and mixed with 4 ml of 50% OptiPrep in a second TH641 20. tube. 2 ml of 5% OptiPrep were overlaid and the sample centrifuged Buffer C: 100 mM sodium phosphate, pH 7.4, 0.15 M NaCl, 4 mM at 52,000 g for 90 minutes at 4°C. The gradient was fractionated in KCl, 2 mM MgCl2, and 0.02% (wt/vol) sodium azide. 0.7 ml fractions. Fraction 3 was designated caveolae membranes (CM). Buffer D: Buffer C containing 1% BSA, 0.05% Tween 20, 0.05% Triton X-100. Immunoblotting Buffer E: Buffer C containing 1% BSA, 0.01% Tween 20, 0.01% Equal amounts of the indicated mouse tissue (40 mg wet mass) was Triton X-100. sonicated in 200 µl 2× SDS-sample buffer (100 mM Tris-HCL, pH Buffer F: 10 mM Tris-HCl pH 7.5, 250 mM sucrose, 0.1 mM 6.8, 20% glycerol, 4% SDS, 4% 2-mercaptoethanol). The 50 µl EDTA. sample was diluted with 950 µl of water before adding 100 µl of 72% TCA to precipitate the protein. The precipitate was redissolved in Cell culture 1000 µl 2× SDS-sample buffer. Equal fractions (30 µl, approx. 300 µg Normal human fibroblasts (Goldstein et al., 1983) grown to wet mass tissue) were heated at 95°C for 5 minutes in SDS-sample confluence in 150 mm plates were cultured in DMEM supplemented buffer (Laemmli, 1970) before being separated by electrophoresis at with penicillin and 10% FBS. 20 hours before each experiment, the 25 mA per gel. The proteins were transferred to PVDF membranes. medium was changed to MEM plus 200 µg/ml of BSA. The pituitary After blocking with buffer B containing 5% nonfat dry milk, the cell line GH3 was cultured (100 mm plates) in F-12K medium membranes were incubated with the first antibody followed by the supplemented with 2.5% FBS and 15% horse serum (HS) for 4 days second antibody conjugated with HRP in buffer B containing 1% before each experiment. nonfat dry milk. Bound antibody was detected using an ECL detection system. To isolate protein from keratinocyte cultures, cell layers were Keratinocyte cell culture washed with PBS and treated for 10 minutes at 4°C with cell lysis Human primary keratinocytes were harvested from healthy adult skin buffer (1.0% NP-40, 20 mM Tris, pH 7.5, 2.0 mM sodium vanadate, obtained as surgical discard tissue.