Identification of minor components of coated vesicles by use of permeation chromatography.

Pfeffer, Suzanne R.; Kelly, Regis B.

doi:10.1083/jcb.91.2.385

Cited by 45 publications

(30 citation statements)

References 22 publications

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“…Our finding that the types of transport vesicles that utilize clathrin are morphologically diverse structures is consistent with the current knowledge of such structures (Pfeffer and Kelly 1981). Consistent with what is known concerning clathrin-coated vesicles, we observed that the typical unit membrane vesicles, which range in size from 50Á150 nM, were coated with clathrin.…”

Section: Discussionsupporting

confidence: 92%

Using quantum dots to visualize clathrin associations

Ogunkoya

Nickel

Murray

2009

Biotechnic & Histochemistry

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Our current understanding of clathrin-mediated endocytosis proposes that the process is initiated at a specialized anatomical structure called a coated pit. Electron microscopy has been required for elucidation of the morphology of coated pits and the vesicles produced therein, and the presence of a bristle coat has been taken as suggestive of clathrin surrounding these vesicles. More recently, immunocytochemical methods have confirmed that endocytic vesicles are surrounded by clathrin and its adaptor proteins, but there is a need to identify precisely and to follow the fate of the cellular organelles seen by fluorescence microscopy. We used quantum immune-electron microscopy to localize clathrin in a human adrenal cortical cell line (SW-13). Clathrin was shown to associate with a variety of vesicle types including the classic clathrin-coated vesicles and pits used in receptor internalization, pentilaminar annular gap junction vesicles, and multivesicular bodies. The images obtained with quantum dot technology allow accurate and specific localization of clathrin and the clathrin adaptor protein, AP-2, with cellular organelles and suggest that some of the structures classified as typical coated vesicles by immunocytochemical light microscopic techniques actually may be membrane bound pits.

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Section: Discussionsupporting

confidence: 92%

Using quantum dots to visualize clathrin associations

Ogunkoya

Nickel

Murray

2009

Biotechnic & Histochemistry

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“…The characterization of these carriers has played a major role in the functional dissection of coat-dependent sorting and vesiculation mechanisms. For example, vesicles "in a basket" isolated from brain led to the biochemical identification of the first sorting machinery, clathrin and the AP-1 and AP-2 adaptors (Kanaseki and Kadota, 1969;Pearse, 1975;Pfeffer and Kelly, 1981;Pearse and Crowther, 1987). Subsequent studies of cargo molecules in brain clathrin-coated vesicles were crucial in revealing mechanisms of synaptic vesicle recycling (Pfeffer and Kelly, 1985;Maycox et al, 1992;Blondeau et al, 2004).…”

Section: Introductionmentioning

confidence: 99%

Phosphatidylinositol-4-Kinase Type II α Is a Component of Adaptor Protein-3-derived Vesicles

Salazar

Craige

Wainer

et al. 2005

MBoC

100

136

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A membrane fraction enriched in vesicles containing the adaptor protein (AP) -3 cargo zinc transporter 3 was generated from PC12 cells and was used to identify new components of these organelles by mass spectrometry. Proteins prominently represented in the fraction included AP-3 subunits, synaptic vesicle proteins, and lysosomal proteins known to be sorted in an AP-3-dependent way or to interact genetically with AP-3. A protein enriched in this fraction was phosphatidylinositol-4-kinase type II␣ (PI4KII␣). Biochemical, pharmacological, and morphological analyses supported the presence of PI4KII␣ in AP-3-positive organelles. Furthermore, the subcellular localization of PI4KII␣ was altered in cells from AP-3-deficient mocha mutant mice. The PI4KII␣ normally present both in perinuclear and peripheral organelles was substantially decreased in the peripheral membranes of AP-3-deficient mocha fibroblasts. In addition, as is the case for other proteins sorted in an AP-3-dependent way, PI4KII␣ content was strongly reduced in nerve terminals of mocha hippocampal mossy fibers. The functional relationship between AP-3 and PI4KII␣ was further explored by PI4KII␣ knockdown experiments. Reduction of the cellular content of PI4KII␣ strongly decreased the punctate distribution of AP-3 observed in PC12 cells. These results indicate that PI4KII␣ is present on AP-3 organelles where it regulates AP-3 function. INTRODUCTIONMembrane-enclosed organelles possess distinctive protein compositions that are dynamically maintained by inbound and outbound vesicle carriers. These vesicles selectively concentrate appropriate membrane proteins while leaving behind resident proteins found in the donor organelle, a process called sorting (Bonifacino and Glick, 2004). Central to membrane protein sorting and vesiculation are a family of cytosolic coat complexes that mediate vesicle budding and function as cargo-specific adaptors. These include monomeric proteins and the heterotetrameric adaptor proteins (AP)-1, -2, -3, and -4 (Bonifacino and Glick, 2004;Robinson, 2004). These coats participate in the generation of vesicles that carry a unique array of membrane protein "cargoes." The characterization of these carriers has played a major role in the functional dissection of coat-dependent sorting and vesiculation mechanisms. For example, vesicles "in a basket" isolated from brain led to the biochemical identification of the first sorting machinery, clathrin and the AP-1 and AP-2 adaptors (Kanaseki and Kadota, 1969;Pearse, 1975;Pfeffer and Kelly, 1981;Pearse and Crowther, 1987). Subsequent studies of cargo molecules in brain clathrin-coated vesicles were crucial in revealing mechanisms of synaptic vesicle recycling (Pfeffer and Kelly, 1985;Maycox et al., 1992;Blondeau et al., 2004). The advent of complete genome sequencing and the concomitant development of informatics tools has led to the rapid identification of proteins by mass spectrometry (Taylor et al., 2003). Application of these methodologies to clathrin-coated vesicles has allowed the identifi...

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“…Since conventional preparations of coated vesicles contain both endocytic and Golgi-derived vesicles, the fact that these fractions have been found to contain ATP-dependent acidification activity does not establish the intracellular origin of the endosomal proton pump. Moreover, since coated vesicle acidification is typically monitored using nonselective externally added pH probes (e.g., acridine orange), interpretation may be further complicated by the presence of small amounts of V-ATPase-containing uncoated membranes in the coated vesicle fraction (7).…”

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confidence: 99%

Rat liver endocytic coated vesicles do not exhibit ATP-dependent acidification in vitro.

Fuchs¹,

Ellinger²,

Pavelka³

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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Coated vesicle fractions from a variety of tissues have been found to contain a vacuolar proton ATPase. Since these fractions contain both plasma membrane-and Golgi-derived coated vesicles, we sought to determine speciftcally whether endocytic coated vesidles from rat liver contain an active vacuolar proton ATPase. Endocytic vesicles (coated vesides and endosomes) were selectively labeled with pHsensitive endocytic tracers (fluorescein isothiocyanate-dextran or -asialoorsomucoid). Coated vesicles were then separated from endosomes by sucrose density gradient centugation. Although the endosomal fractions were found to exhibit significant ATP-dependent acidification activity, highly purified coated vesidles conining pH-senstive endocytic tracers were unable to generate a pH gradient in response to ATP addition. The coated vesicles could be passively acidified, however, by creating potassium diffson potentials, indicating that they were in fact capable of mntaining proton gradients. Moreover, signcant ATP-dependent acidification acvity was observed when the coated vedsce fractions were assayed using the nonselective externally added pH probe acridine orange. Thus, it appears that rat liver endocytic coated vesicles do not contain a functional proton pump. The active vacuolar proton ATlase found in these fractions instead reflected the presence of Golgi-derived coated vesicles or contaminating membranes.Many intracellular organelles of the endocytic and biosynthetic pathways maintain an acidic internal pH due to the activity of a vacuolar proton ATPase (V-ATPase). The main characteristics of this multisubunit V-ATPase are its electrogenicity and specific inhibition by N-ethylmaleimide and bafilomycin (1). Thus far, coated vesicles, endosomes, lysosomes, trans-Golgi elements, endocrine secretory granules, and cholinergic synaptic vesicles have been shown to be acidic (2, 3). Since these organelles maintain distinct pH values in vivo, their capacities for ATP-driven acidification must be regulated. While acidification in vitro has been shown to be regulated by the membrane potential (1, 3, 4), it is also possible that pH regulation is also controlled by regulating the activity or intracellular targeting of the V-ATPase itself.This raises the issue how functional proton pumps are delivered to endosomes and lysosomes. The most common view is that coated vesicles, which have long been known to possess a V-ATPase (5, 6), internalize proton pumps from the plasma membrane and deliver them to early endosomes and, thus, to late endosomes and lysosomes. It is possible, however, that V-ATPases are targeted to endosomes from the biosynthetic pathway via Golgi-derived coated vesicles. Since conventional preparations of coated vesicles contain both endocytic and Golgi-derived vesicles, the fact that these fractions have been found to contain ATP-dependent acidification activity does not establish the intracellular origin of the endosomal proton pump. Moreover, since coated vesicle acidification is typically monitored using...

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Identification of minor components of coated vesicles by use of permeation chromatography.

Cited by 45 publications

References 22 publications

Using quantum dots to visualize clathrin associations

Using quantum dots to visualize clathrin associations

Phosphatidylinositol-4-Kinase Type II α Is a Component of Adaptor Protein-3-derived Vesicles

Rat liver endocytic coated vesicles do not exhibit ATP-dependent acidification in vitro.

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