αTAT1 catalyses microtubule acetylation at clathrin-coated pits.

In most eukaryotic cells microtubules undergo post-translational modifications such as acetylation of α-tubulin on lysine 40, a widespread modification restricted to a subset of microtubules that turns over slowly. This subset of stable microtubules accumulates in cell protrusions and regulates cell polarization, migration and invasion. However, mechanisms restricting acetylation to these microtubules are unknown. Here we report that clathrin-coated pits (CCPs) control microtubule acetylation through a direct interaction of the α-tubulin acetyltransferase αTAT1 (refs 8, 9) with the clathrin adaptor AP2. We observe that about one-third of growing microtubule ends contact and pause at CCPs and that loss of CCPs decreases lysine 40 acetylation levels. We show that αTAT1 localizes to CCPs through a direct interaction with AP2 that is required for microtubule acetylation. In migrating cells, the polarized orientation of acetylated microtubules correlates with CCP accumulation at the leading edge, and interaction of αTAT1 with AP2 is required for directional migration. We conclude that microtubules contacting CCPs become acetylated by αTAT1. In migrating cells, this mechanism ensures the acetylation of microtubules oriented towards the leading edge, thus promoting directional cell locomotion and chemotaxis.

Oligomerized Tie2 localizes to clathrin-coated pits in response to angiopoietin-1.

The tyrosine kinase receptor Tie2 is expressed on endothelial cells, and together with its ligand angiopoietin-1 (Ang1), is important for angiogenesis and vascular stability. Upon activation by Ang1, Tie2 is rapidly internalized and degraded, a mechanism most likely necessary to attenuate receptor activity. Using immunogold electron microscopy, we show that on the surface of endothelial cells, Tie2 is arranged in variably sized clusters containing dimers and higher order oligomers. Clusters of Tie2 were expressed on the apical and basolateral plasma membranes, and on the tips of microvilli. Upon activation by Ang1, Tie2 co-localized with the clathrin heavy chain at the apical and basolateral plasma membranes and within endothelial cells indicating that Tie2 internalizes through clathrin-coated pits. Inhibiting cellular endocytosis by depleting cellular potassium or by acidifying the cytosol blocked the internalization of Tie2 in response to Ang1. Our results suggest that one pathway mediating the internalization of Tie2 in response to Ang1 is through clathrin-coated pits.

Endocytic accessory proteins are functionally distinguished by their differential effects on the maturation of clathrin-coated pits.

Diverse cargo molecules (i.e., receptors and ligand/receptor complexes) are taken into the cell by clathrin-mediated endocytosis (CME) utilizing a core machinery consisting of cargo-specific adaptors, clathrin and the GTPase dynamin. Numerous endocytic accessory proteins are also required, but their differential roles and functional hierarchy during CME are not yet understood. Here, we used a combination of quantitative live-cell imaging by total internal reflection fluorescence microscopy (TIR-FM), and decomposition of the lifetime distributions of clathrin-coated pits (CCPs) to measure independent aspects of CCP dynamics, including the turnover of abortive and productive CCP species and their relative contributions. Capitalizing on the sensitivity of this assay, we have examined the effects of specific siRNA-mediated depletion of endocytic accessory proteins on CME progression. Of the 12 endocytic accessory proteins examined, we observed seven qualitatively different phenotypes upon protein depletion. From this data we derive a temporal hierarchy of protein function during early steps of CME. Our results support the idea that a subset of accessory proteins, which mediate coat assembly, membrane curvature, and cargo selection, can provide input into an endocytic restriction point/checkpoint mechanism that monitors CCP maturation.

AAK1 regulates Numb function at an early step in clathrin-mediated endocytosis.

Numb is an endocytic protein that is proposed to influence clathrin-coated pit assembly, although its mode of action and the mechanisms that regulate its activity are unknown. In this study, we show that Numb binds to and is phosphorylated by adaptor-associated kinase 1 (AAK1), a key endocytic kinase. We find that AAK1 redistributes Numb to perinuclear endosomes when overexpressed, while kinase depletion causes Numb to accumulate at the plasma membrane. Overexpression of a Numb point mutant (T102A) that lacks the AAK1 phosphorylation site potently disrupts transferrin and low-density lipoprotein internalization but does not impact EGF uptake. Consistent with Numb redistribution results, we find that T102A Numb no longer localizes to perinuclear endosomes. Instead, it is enriched at the plasma membrane where it shows elevated levels of colocalization with coated pit markers. Collectively, these observations demonstrate that Numb endocytic activity is regulated by AAK1 and that phosphorylation may be a critical step in promoting coated pit maturation.

The class II phosphoinositide 3-kinase PI3K-C2alpha is concentrated in the trans-Golgi network and present in clathrin-coated vesicles.

In recent years, a large family of phosphoinositide 3-kinase (PI3K) isozymes has been characterized and cloned. Several of these PI3K enzymes have overlapping tissue distributions and it remains unclear if and how their 3-phosphoinositide products elicit differential, intracellular effects. One possibility is that the PI3K enzymes display a restricted distribution within the cell to produce their 3-phospholipid products in specific, subcellular compartments. In the present study we characterize the subcellular distribution of the novel class II PI3K isozyme PI3K-C2alpha in several mammalian cell types. Differential centrifugation of COS-1 and U937 cells together with Western blot analysis demonstrated that PI3K-C2alpha is constitutively associated with phospholipid membranes. Centrifugation of rat brain homogenates and Western blotting revealed that in contrast to the class IA PI3K enzymes, PI3K-C2alpha could be co-purified with a population of clathrin-coated vesicles (CCVs). Furthermore, a PI3K activity refractory to wortmannin treatment was detected in CCV preparations consistent with the presence of the PI3K-C2alpha isozyme. These biochemical observations were supported by immunofluorescence analysis that revealed PI3K-C2alpha to have a punctate distribution and an enrichment of immunoreactivity within a perinuclear site consistent with its presence in the endoplasmic reticulum or Golgi apparatus. Dual label immunofluorescence demonstrated that in this region, the distribution of PI3K-C2alpha closely paralleled that of gamma-adaptin, a component of the AP-1 adaptor that is present in the trans-Golgi and the trans-Golgi network (TGN) resident protein TGN-46. Neither the phospholipid association nor the subcellular localization of PI3K-C2alpha was dependent upon either its COOH-terminal PX or C2 domains. Mutants lacking these domains demonstrated a similar distribution to the wild type enzyme when expressed as recombinant proteins. Treatment of cells with brefeldin A disrupted the perinuclear staining pattern of both PI3K-C2alpha and the AP-1 complex demonstrating that the localization of both molecules at the TGN is dependent upon ADP-ribosylation factor GTPase activity.

Hepatic sinusoidal endothelial fenestrae express plasma membrane Ca++pump and Ca++Mg++-ATPase.

BACKGROUND/AIM: In general, intracytoplasmic free calcium ions (Ca++) are maintained at a very low concentration in mammalian tissue by extruding Ca++ against a high concentration of extracellular Ca++, mainly through the activity of the plasma membrane Ca++pump-ATPase. The aim of the present study was to demonstrate by electron cytochemical and immunogold methods the ultrastructural localization of two different types of plasma membrane Ca++-ATPase, i.e. Ca++Mg++-ATPase and Ca++pump-ATPase in the hepatic sinusoidal endothelium. METHODS: Liver tissues and the isolated hepatic sinusoidal endothelial cell (SEC)s were subjected to the following procedures. The ultrastructural localizations of Ca++Mg++-ATPase were examined by an electron cytochemical method. The ultrastructural localization of Ca++pump-ATPase was identified by an electron immunogold method. RESULTS: The cytochemical reaction of Ca++Mg++-ATPase was found to be localized on the outer sites of the plasma membrane of sinusoidal endothelial fenestrae (SEF). The immunogold particles indicating the presence of Ca++pump-ATPase were identified on the inner sites (cytoplasmic) of the invaginated plasma membrane of SEF CONCLUSIONS: Both Ca++Mg++-ATPase and Ca++pump-ATPase demonstrated on the SEF plasma membrane may be involved in the regulation of intracytoplasmic Ca++ concentration.

Structure, mechanism and regulation of the clathrin-coated vesicle and yeast vacuolar H(+)-ATPases.

The vacuolar H(+)-ATPases (or V-ATPases) are a family of ATP-dependent proton pumps that carry out acidification of intracellular compartments in eukaryotic cells. This review is focused on our work on the V-ATPases of clathrin-coated vesicles and yeast vacuoles. The coated-vesicle V-ATPase undergoes trafficking to endosomes and synaptic vesicles, where it functions in receptor recycling and neurotransmitter uptake, respectively. The yeast V-ATPase functions to acidify the central vacuole and is necessary both for protein degradation and for coupled transport processes across the vacuolar membrane. The V-ATPases are multisubunit complexes composed of two functional domains. The V(1) domain is a 570 kDa peripheral complex composed of eight subunits of molecular mass 73-14 kDa (subunits A-H) that is responsible for ATP hydrolysis. The V(o) domain is a 260 kDa integral complex composed of five subunits of molecular mass 100-17 kDa (subunits a, d, c, c' and c") that is responsible for proton translocation. To explore the function of individual subunits in the V-ATPase complex as well as to identify residues important in proton transport and ATP hydrolysis, we have employed a combination of chemical modification, site-directed mutagenesis and in vitro reassembly. A central question concerns the mechanism by which vacuolar acidification is controlled in eukaryotic cells. We have proposed that disulfide bond formation between conserved cysteine residues at the catalytic site of the V-ATPase plays an important role in regulating V-ATPase activity in vivo. Other regulatory mechanisms that are discussed include reversible dissociation and reassembly of the V-ATPase complex, changes in the tightness of coupling between proton transport and ATP hydrolysis, differential targeting of V-ATPases within the cell and control of the Cl(-) conductance that is necessary for vacuolar acidification.

Structure of the vacuolar ATPase by electron microscopy.

The structure of the vacuolar ATPase from bovine brain clathrin-coated vesicles has been determined by electron microscopy of negatively stained, detergent-solubilized enzyme molecules. Preparations of both lipid-containing and delipidated enzyme have been analyzed. The complex is organized in two major domains, a V(1) and V(0), with overall dimensions of 28 x 14 x 14 nm. The V(1) is a more or less spherical molecule with a central cavity. The V(0) has the shape of a flattened sphere or doughnut with a radius of about 100 A. The V(1) and V(0) are joined by a 60-A long and 40-A wide central stalk, consisting of several individual protein densities. Two kinds of smaller densities are visible at the top periphery of the V(1), and one of these seems to extend all the way down to the stalk domain in some averages. Images of both the lipid-containing and the delipidated complex show a 30-50-kDa protein density on the lumenal side of the complex, opposite the central stalk, centered in the ring of c subunits. A large trans-membrane mass, probably the C-terminal domain of the 100-kDa subunit a, is seen at the periphery of the c subunit ring in some projections. This large mass has both a lumenal and a cytosolic domain, and it is the cytosolic domain that interacts with the central stalk. Two to three additional protein densities can be seen in the V(1)-V(0) interface, all connected to the central stalk. Overall, the structure of the V-ATPase is similar to the structure of the related F(1)F(0)-ATP synthase, confirming their common origin.

Interaction of the clathrin-coated vesicle V-ATPase with ADP and sodium azide.

The kinetics of adenosine triphosphate (ATP)-dependent proton transport into clathrin-coated vesicles from bovine brain have been studied. We observe that the vacuolar proton-translocating ATPase (V-ATPase) from clathrin-coated vesicles is subject to two different types of inhibition by ADP. The first is competitive inhibition with respect to ATP, with a Ki for ADP of 11 microM. The second type of inhibition occurs after preincubation of the V-ATPase in the presence of ADP and Mg2+, which results in inhibition of the initial rate of proton transport followed by reactivation over the course of several minutes. The second effect is observed at ADP concentrations as low as 0.1-0.2 microM, indicating that a high affinity inhibitory complex is formed between ADP and the V-ATPase and is only slowly dissociated after the addition of ATP. We have further investigated the effect of sodium azide, an inhibitor of the F-ATPases that has been shown to stabilize an inactive complex between ADP and the F1-F0-ATP synthase (F-ATPase). We observed that azide inhibited ATP-dependent proton transport by the purified, reconstituted V-ATPase with a K0.5 of 0.2-0.4 mM but had no effect on ATP hydrolysis. Azide was shown not to increase the passive proton permeability of reconstituted vesicles and did not stimulate ATP hydrolysis by the reconstituted enzyme, in contrast with CCCP, which both abolished the proton gradient and stimulated hydrolysis. Thus, azide does not appear to act as a simple uncoupler of proton transport and ATP hydrolysis. Rather, azide may have some more direct effect on V-ATPase activity. Possible mechanisms by which azide could exert this effect on the V-ATPase and the contrasting effects of azide on the F- and V-ATPases are discussed.

Ubiquitously expressed dynamin-II has a higher intrinsic GTPase activity and a greater propensity for self-assembly than neuronal dynamin-I.

To begin to understand mechanistic differences in endocytosis in neurons and nonneuronal cells, we have compared the biochemical properties of the ubiquitously expressed dynamin-II isoform with those of neuron-specific dynamin-I. Like dynamin-I, dynamin-II is specifically localized to and highly concentrated in coated pits on the plasma membrane and can assemble in vitro into rings and helical arrays. As expected, the two closely related isoforms share a similar mechanism for GTP hydrolysis: both are stimulated in vitro by self-assembly and by interaction with microtubules or the SH3 domain-containing protein, grb2. Deletion of the C-terminal proline/arginine-rich domain from either isoform abrogates self-assembly and assembly-dependent increases in GTP hydrolysis. However, dynamin-II exhibits a approximately threefold higher rate of intrinsic GTP hydrolysis and higher affinity for GTP than dynamin-I. Strikingly, the stimulated GTPase activity of dynamin-II can be >40-fold higher than dynamin-I, due principally to its greater propensity for self-assembly and the increased resistance of assembled dynamin-II to GTP-triggered disassembly. These results are consistent with the hypothesis that self-assembly is a major regulator of dynamin GTPase activity and that the intrinsic rate of GTP hydrolysis reflects a dynamic, GTP-dependent equilibrium of assembly and disassembly.

Receptor-mediated inhibition of renal Na(+)-K(+)-ATPase is associated with endocytosis of its alpha- and beta-subunits.

The mechanisms involved in receptor-mediated inhibition of Na(+)-K(+)-ATPase remain poorly understood. In this study, we evaluate whether inhibition of proximal tubule Na(+)-K(+)-ATPase activity by dopamine is linked to its removal from the plasma membrane and internalization into defined intracellular compartments. Clathrin-coated vesicles were isolated by sucrose gradient centrifugation and negative lectin selection, and early and late endosomes were separated on a flotation gradient. Inhibition of Na(+)-K(+)-ATPase activity by dopamine, in contrast to its inhibition by ouabain, was accompanied by a sequential increase in the abundance of the alpha-subunit in clathrin-coated vesicles (1 min), early endosomes (2.5 min), and late endosomes (5 min), suggesting its stepwise translocation between these organelles. A similar pattern was found for the beta-subunit. The increased incorporation of both subunits in all compartments was blocked by calphostin C. The results demonstrate that the dopamine-induced decrease in Na(+)-K(+)-ATPase activity in proximal tubules is associated with internalization of its alpha- and beta-subunits into early and late endosomes via a clathrin-dependent pathway and that this process is protein kinase C dependent. The presence of Na(+)-K(+)-ATPase subunits in endosomes suggests that these compartments may constitute normal traffic reservoirs during pump degradation and/or synthesis.

The Src family kinases, Fgr, Fyn, Lck, and Lyn, colocalize with coated membranes in platelets.

Nonreceptor protein tyrosine kinases phosphorylate proteins, thereby activating many intracellular signaling pathways and mediating protein-protein interactions. Protein phosphorylation is regulated in large part by the subcellular localization of these kinases and their respective substrates. Src is the most studied of these kinases, although other members of the Src family have been shown to be important in the differentiation of specific cell types. Src and Src family members are reported to be membrane-associated, but detergent-extraction studies have demonstrated a major difference in the solubility of Src compared with other members of the Src family (Fgr, Fyn, Lck, Lyn, and Yes), suggesting that their subcellular distributions may be different. By immunoelectron microscopy, we demonstrate that, unlike Src, the Src-related kinases are associated with electron-dense cytoplasmic domains and plasma membrane domains that correspond in size and frequency to endocytotic vesicles and coated pits. Clusters of labeling for these kinases also were seen adjacent to granule membranes. These kinases colocalize with the coated vesicle protein, clathrin, confirming their association with this class of endocytotic vesicle. We hypothesize that this vesicular association of Src-related kinases indicates a role for them in the endocytotic vesicle-mediated uptake and trafficking of plasma proteins into platelet granules.

Dynamin GTPase, a force-generating molecular switch.

Dynamin is a GTPase that regulates late events in clathrin-coated vesicle formation. Our current working model suggests that dynamin is targeted to coated pits in its unoccupied or GDP-bound form, where it is initially distributed uniformly throughout the clathrin lattice. GTP/GDP exchange triggers its release from these sites and its assembly into short helices that encircle the necks of invaginated coated pits like a collar. GTP hydrolysis, which is required for vesicle detachment, presumably induces a concerted conformation change, tightening the collar. Unlike most of its GTPase cousins that serve as molecular switches, dynamin has a low affinity for GTP, a very high intrinsic rate of GTP hydrolysis and functions as a homo-oligomer. A concerted conformational change resulting from coordinated GTP hydrolysis by the dynamin oligomer might be sufficient to generate force. In this case, dynamin would be the first GTPase identified that acts as a structural protein with mechano-chemical function.

Identification of a 14-kDa subunit associated with the catalytic sector of clathrin-coated vesicle H+-ATPase.

The clathrin-coated vesicle H+-ATPase is composed of a peripheral catalytic sector (VC) and an integral membrane proton channel (VB), both of which are multiple subunit complexes. This study was conducted to determine if subunit F, previously identified in vacuolar proton pumps of tobacco hornworm and yeast, was present in mammalian pumps. Using a polymerase chain reaction-based strategy, we have isolated and sequenced cDNA clones from bovine and rat brain cDNA libraries. A full-length clone from rat brain encodes a 119-amino acid polypeptide with a predicted molecular mass of 13, 370 Da and with approximately 72 and 49% identity to subunit F of tobacco hornworm and yeast, respectively. Southern and Northern blot analyses indicate that the protein is encoded by a single gene. An anti-peptide antibody, directed against deduced protein sequence, was affinity-purified and shown to react with a 14-kDa polypeptide that is present in a highly purified pump prepared from clathrin-coated vesicles and also isolated VC. When stripped clathrin-coated vacuolars and purified chromaffin granule membranes were treated with KI in the presence of ATP, the 14-kDa subunit was released from both membranes, further indicating that it is part of the peripheral catalytic sector. In addition, direct sequencing of this 14-kDa component of the coated vacuolar proton pump confirmed its identity as a subunit F homologue.

Comparison of the coated-vesicle and synaptic-vesicle vacuolar (H+)-ATPases.

The V-ATPases are a novel class of ATP-dependent proton pumps responsible for acidification of intracellular compartments in eukaryotic cells. They play an important role in receptor-mediated endocytosis, intracellular membrane traffic, macromolecular processing and degradation and coupled transport, as well as functioning in the plasma membrane of certain specialized cell types. The V-ATPases are multisubunit complexes that are organized into a peripheral V1 complex responsible for ATP hydrolysis and an integral V0 domain responsible for proton translocation. Regulation of vacuolar acidification is critical to its role in membrane traffic and other cellular processes. We are currently investigating several mechanisms of regulation of vacuolar acidification, including disulfide bond formation between cysteine residues located at the catalytic site, control of assembly of the peripheral and integral domains, and differential targeting of V-ATPases to different intracellular destinations using their interaction with organelle-specific adaptin complexes.

Reconstitution of recombinant 33-kDa subunit of the clathrin-coated vesicle H(+)-ATPase.

Evidence suggests that the ATP hydrolytic sector of the clathrin-coated vesicle proton-translocating ATPase is composed of four subunits of molecular masses of 70, 58, 40, and 33 kDa (Xie, X. S., and Stone, D. K. (1988) J. Biol. Chem. 263, 9859-9867). We have now expressed recombinant 33-kDa polypeptide in Escherichia coli and in Spodoptera frugiperda (Sf9) cells. This subunit, renatured and purified from both sources, lacks intrinsic ATPase activity. Co-reconstitution of these recombinant 33-kDa polypeptides and recombinant 40-kDa subunit to a biochemically prepared 70-58-kDa subcomplex results in a 6-fold stimulation of calcium-activated, N-ethyl-maleimide-sensitive ATPase activity, documenting the essential role of the 33- and 40-kDa components in vacuolar type proton pump function and furthering the aim of reconstitution of a purely recombinant hydrolytic core.

Reconstitution of recombinant 40-kDa subunit of the clathrin-coated vesicle H(+)-ATPase.

We have proposed a model of the ATP hydrolytic sector of the clathrin-coated vesicle H(+)-ATPase wherein significant catalysis requires four subunits of molecular masses of 70, 58, 40, and 33 kDa (Xie, X.-S., and Stone, D. K. (1988) J. Biol. Chem. 263, 9859-9867). We have cloned and expressed the 40-kDa component in Escherichia coli and have purified the recombinant protein to homogeneity. This subunit lacks ATP hydrolytic capacity, but when reconstituted to a 40 kDa-depleted hydrolytic sector, there is a greater than 20-fold increase in calcium-activated, N-ethylmaleimide-sensitive ATP hydrolysis, indicating that this subunit is required for vacuolar-type proton pump function.

NADH-ascorbate free radical and -ferricyanide reductase activities represent different levels of plasma membrane electron transport.

Plasma membranes isolated from rat liver by two-phase partition exhibited dehydrogenase activities for ascorbate free radical (AFR) and ferricyanide reduction in a ratio of specific activities of 1:40. NADH-AFR reductase could not be solubilized by detergents from plasma membrane fractions. NADH-AFR reductase was inhibited in both clathrin-depleted membrane and membranes incubated with anti-clathrin antiserum. This activity was reconstituted in plasma membranes in proportion to the amount of clathrin-enriched supernatant added. NADH ferricyanide reductase was unaffected by both clathrin-depletion and antibody incubation and was fully solubilized by detergents. Also, wheat germ agglutinin only inhibited NADH-AFR reductase. The findings suggest that NADH-AFR reductase and NADH-ferricyanide reductase activities of plasma membrane represent different levels of the electron transport chain. The inability of the NADH-AFR reductase to survive detergent solubilization might indicate the involvement of more than one protein in the electron transport from NADH to the AFR but not to ferricyanide.

The coated vesicle vacuolar (H+)-ATPase associates with and is phosphorylated by the 50-kDa polypeptide of the clathrin assembly protein AP-2.

We have previously noted a 50-kDa polypeptide (p50) co-purifying with preparations of the bovine brain clathrin-coated vesicle vacuolar (H+)-ATPase (V-ATPase) (Zhang, J., Myers, M., and Forgac, M. (1992) J. Biol. Chem. 267, 9773-9778). We show that p50 is also immunoprecipitated with the V-ATPase, further suggesting its specific association with the proton pump. To determine the identity of this 50-kDa polypeptide and the stoichiometry of its association with the V-ATPase, we performed N-terminal amino acid sequencing and quantitative amino acid analysis of the gel-purified protein. These results revealed the unknown polypeptide to be the 50-kDa subunit of the clathrin assembly protein AP-2 (AP50); we estimate the stoichiometry of association is one AP50 per V-ATPase complex. AP50 is an N-ethylmaleimide (NEM)-inhibitable autokinase and incubation of purified V-ATPase with [gamma-32P]ATP resulted in the NEM-sensitive phosphorylation of AP50 and the B subunit of the V-ATPase. The same phosphorylation pattern is seen if the labeling reaction is done with intact clathrin-coated vesicles and the V-ATPase subsequently immunoprecipitated from the solubilized vesicles. This represents the first report of phosphorylation of one of the V-ATPase subunits. The functional significance of this phosphorylation for regulation or targeting of the V-ATPase in vivo remains to be determined.

Identification of membrane-bound carbonic anhydrase in white matter coated vesicles: the fate of carbonic anhydrase and other white matter coated vesicle proteins in triethyl tin-induced leukoencephalopathy.

We have extended our studies on the content of white matter derived coated vesicles (WMCVs) to show that they are enriched in membrane-bound carbonic anhydrase. Within the myelin complex membrane-bound carbonic anhydrase is concentrated in the periaxolemmal domain; however, this protein is enriched almost sevenfold in the bilayer of coated vesicles even relative to this myelin membrane region. These data suggest that some vesicles are derived from a site at which this enzyme is highly localized. The enrichment observed for membrane-bound carbonic anhydrase is unique since other periaxolemmal proteins such as CNPase and plasmolipin are only present in equal amounts in periaxolemmal-myelin fractions and WMCVs. Based on their known localization, the presence of CNPase coupled with the absence of MAG in WMCVs suggest that these vesicles are derived from the paranodal region. The identification in WMCVs of periaxolemmal-myelin proteins associated with ion and fluid movement, such as carbonic anhydrase, Na+,K+ ATPase, and the putative K+ channel protein plasmolipin, prompted us to examine the status of these vesicles in triethyl tin (TET)-induced myelin edema. Coated vesicles and other membrane fractions were isolated from whole brains of control and TET-treated rats. Whole brains were used so we could compare the effects of TET on WMCV proteins with the effect on proteins enriched in gray matter coated vesicles. The results indicated that TET had no detectable effect on compact or periaxolemmal-myelin, however, Western blot analysis showed that WMCV proteins, such as carbonic anhydrase, CNPase, and plasmolipin, were virtually absent or greatly diminished from the whole brain coated vesicle fraction.(ABSTRACT TRUNCATED AT 250 WORDS)