Isolation of Clathrin‐Coated Vesicles by Differential and Density Gradient Centrifugation

Abstract

Endocytosis of nutrients, signaling receptors, and other cell surface regulatory proteins via clathrin-coated vesicles (CCVs) is essential for normal cellular function. CCVs are also involved in the transport of proteins, such as lysosomal hydrolases, from the trans-Golgi network to the endosomal/lysosomal system. CCVs are relatively uniform in size and, at 50 to 100 nm in diameter, they are among the smallest membranous organelles. Moreover, they are encased in a dense, proteinaceous coat, which contributes to the formation of the vesicular structure. Protocols for isolating CCVs take advantage of the small size and high density of these organelles by using differential centrifugation coupled with velocity and equilibrium gradients to separate CCVs from contaminating membranes.The first reliable protocol for isolating CCVs was described by Pearse (1975), working with pig brain. CCVs were enriched in a microsomal fraction prepared by differential centrifugation. They were subsequently separated from larger and less dense membranes using velocity and equilibrium sedimentation, respectively, on linear sucrose gradients. Pearse (1982) later introduced a protocol (for purification of CCVs from human placenta) in which Ficoll and D 2 O were substituted for sucrose. Most protocols currently in use are derived from these original protocols.In Basic Protocol 1, the authors describe a procedure for isolating CCVs from adult rat brain. This procedure, which is based on the protocol of Maycox et al. (1992), uses differential centrifugation coupled with Ficoll and D 2 O-sucrose density gradient centrifugation. The application of an additional step involving velocity sedimentation in linear sucrose gradients, as originally described by Wasiak et al. (2002), is also outlined. In Alternate Protocols 1 and 2, the authors describe how the same basic approach can be applied to the isolation of CCVs from developing rat brain and cell lines, respectively.When applied to other tissues, such as rat liver, the steps outlined in Basic Protocol 1 yield CCV preparations that exhibit only modest enrichment and purity. In Basic Protocol 2, therefore, the authors describe a fractionation procedure that has been used to purify CCVs from rat liver. This protocol, based on that of Pilch et al. (1983), involves differential centrifugation coupled with velocity and equilibrium centrifugation using discontinuous sucrose gradients.Following fractionation, it is necessary to characterize CCV enrichment and purity. One important method for doing so is the analysis of equal protein aliquots of the various subcellular fractions by SDS-PAGE (UNIT 6.1) using Coomassie blue staining and/or immunoblotting (UNIT 6.2) with antibodies against specific CCV proteins (e.g., clathrin). Thus, for each protocol, we indicate fractions from which aliquots should be retained for analysis. The purity of CCVs is best assessed by electron microscopy (EM). In the Support Protocol, we describe an EM procedure, based on that of Baudhuin et al. (1967), that involves filtrati...

Disabled-2 Protein Facilitates Assembly Polypeptide-2-independent Recruitment of Cystic Fibrosis Transmembrane Conductance Regulator to Endocytic Vesicles in Polarized Human Airway Epithelial Cells

“…Density Gradient Separation and Differential Centrifugation of CCV-To isolate CCVs, subcellular fractionation was performed by density gradient and differential centrifugation as described previously (28). After washing with ice-cold PBS at 4°C, CFBE41o-cells were scraped in buffer A, pH 6.5, containing 1 M MES, 10 mM EGTA, and 0.5 M MgCl 2 and homogenized in a glass-Teflon homogenizer using 20 strokes at 1500 rpm.…”

Non-stoichiometric Relationship between Clathrin Heavy and Light Chains Revealed by Quantitative Comparative Proteomics of Clathrin-coated Vesicles from Brain and Liver

“…Preparation and Analysis of Liver CCVs-Liver CCVs were isolated using procedures described previously (21,22) from adult rats that had been starved overnight. Suspensions of liver CCVs were deposited on 0.22-m nitrocellulose filters to ensure random sampling (23) and were then processed for electron microscopy (EM) using an osmium tetroxide and tannic acid double fixation procedure (24).…”

“…Other Subcellular Fractionation Procedures-CCVs were isolated from adult rat brains as described previously (15,22). For specific experiments, CCVs were isolated from the livers of starved adult rats using the same protocol.…”

Interplay between N-WASP and CK2 optimizes clathrin-mediated endocytosis of EGFR

“…To release the temperature block and concomitantly induce EGFR internalization, this medium was replaced by pre-warmed medium supplemented with EGF (100 ng/ml) and cells were incubated at 37°C. After the indicated time, plates were cooled on ice and all subsequent steps were performed on ice (Girard et al, 2005). Cells were washed three times with ice-cold F-F-54-30-II Buffer A (0.1 M MES pH 6.5, 1 mM EGTA, 0.5 mM MgCl 2 , 1 mM PMSF, 1 g/ml aprotinin, 1 g/ml leupeptin, 1 M pepstatin A, 1 mM DTT, 1 mM sodium orthovanadate and 5 mM NaF), scraped with a rubber policeman into Buffer A and dounce-homogenized (40 strokes with the tight pestle).…”