A comparative study of fixation techniques in the open Bankart operation using either a cannulated screw or suture-anchors

Neuroendocrine PC12 cells contain small microvesicles that closely resemble synaptic vesicles in their physical and chemical properties. Two defining characteristics of synaptic vesicles are their homogeneous size and their unique protein composition. Since synaptic vesicles arise by endocytosis from the plasma membrane, nerve terminals and PC12 cells must contain the molecular machinery to sort synaptic vesicles from other membrane proteins and pinch off vesicles of the correct diameter from a precursor compartment. A cell-free reconstitution system was developed that generates vesicles from PC12 membrane precursors in the presence of ATP and brain cytosol and is temperature dependent. At 15 degrees C, surface-labeled synaptic vesicle proteins accumulate in a donor compartment, while labeled synaptic vesicles cannot be detected. The block of synaptic vesicle formation at 15 degrees C enables the use of the monoclonal antibody, KT3, a specific marker for the epitope-tagged synaptic vesicle protein, VAMP-TAg, to label precursors in the synaptic vesicle biogenesis pathway. From membranes labeled in vivo at 15 degrees C, vesicles generated in vitro at 37 degreesC had the sedimentation characteristics of neuroendocrine synaptic vesicles on glycerol velocity gradients, and excluded the transferrin receptor. Therefore, vesiculation and sorting can be studied in this cell-free system.     

Synaptic-like microvesicles of neuroendocrine cells originate from a novel compartment that is continuous with the plasma membrane and devoid of transferrin receptor

We have characterized the compartment from which synaptic-like microvesicles (SLMVs), the neuroendocrine counterpart of neuronal synaptic vesicles, originate. For this purpose we have exploited the previous observation that newly synthesized synaptophysin, a membrane marker of synaptic vesicles and SLMVs, is delivered to the latter organelles via the plasma membrane and an internal compartment. Specifically, synaptophysin was labeled by cell surface biotinylation of unstimulated PC12 cells at 18 degrees C, a condition which blocked the appearance of biotinylated synaptophysin in SLMVs and in which there appeared to be no significant exocytosis of SLMVs. The majority of synaptophysin labeled at 18 degrees C with the membrane-impermeant, cleavable sulfo-NHS-SS-biotin was still accessible to extracellularly added MesNa, a 150-D membrane-impermeant thiol-reducing agent, but not to the 68,000-D protein avidin. The SLMVs generated upon reversal of the temperature to 37 degrees C originated exclusively from the membranes containing the MesNa-accessible rather than the MesNa-protected population of synaptophysin molecules. Biogenesis of SLMVs from MesNa-accessible membranes was also observed after a short (2 min) biotinylation of synaptophysin at 37 degrees C followed by chase. In contrast to synaptophysin, transferrin receptor biotinylated at 18 degrees or 37 degrees C became rapidly inaccessible to MesNa. Immunofluorescence and immunogold electron microscopy of PC12 cells revealed, in addition to the previously described perinuclear endosome in which synaptophysin and transferrin receptor are colocalized, a sub-plasmalemmal tubulocisternal membrane system distinct from caveolin-positive caveolae that contained synaptophysin but little, if any, transferrin receptor. The latter synaptophysin was selectively visualized upon digitonin permeabilization and quantitatively extracted, despite paraformaldehyde fixation, by Triton X-100. Synaptophysin biotinylated at 18 degrees C was present in these subplasmalemmal membranes. We conclude that SLMVs originate from a novel compartment that is connected to the plasma membrane via a narrow membrane continuity and lacks transferrin receptor.     

GLUT4 and transferrin receptor are differentially sorted along the endocytic pathway in CHO cells

The trafficking of GLUT4, a facilitative glucose transporter, is examined in transfected CHO cells. In previous work, we expressed GLUT4 in neuroendocrine cells and fibroblasts and found that it was targeted to a population of small vesicles slightly larger than synaptic vesicles (Herman, G.A, F. Bonzelius, A.M. Cieutat, and R.B. Kelly. 1994. Proc. Natl. Acad. Sci. USA. 91: 12750-12754.). In this study, we demonstrate that at 37 degrees C, GLUT4-containing small vesicles (GSVs) are detected after cell surface radiolabeling of GLUT4 whereas uptake of radioiodinated human transferrin does not show appreciable accumulation within these small vesicles. Immunofluorescence microscopy experiments show that at 37 degrees C, cell surface-labeled GLUT4 as well as transferrin is internalized into peripheral and perinuclear structures. At 15 degrees C, endocytosis of GLUT4 continues to occur at a slowed rate, but whereas fluorescently labeled GLUT4 is seen to accumulate within large peripheral endosomes, no perinuclear structures are labeled, and no radiolabeled GSVs are detectable. Shifting cells to 37 degrees C after accumulating labeled GLUT4 at 15 degrees C results in the reappearance of GLUT4 in perinuclear structures and GSV reformation. Cytosol acidification or treatment with hypertonic media containing sucrose prevents the exit of GLUT4 from peripheral endosomes as well as GSV formation, suggesting that coat proteins may be involved in the endocytic trafficking of GLUT4. In contrast, at 15 degrees C, transferrin continues to traffic to perinuclear structures and overall labels structures similar in distribution to those observed at 37 degrees C. Furthermore, treatment with hypertonic media has no apparent effect on transferrin trafficking from peripheral endosomes. Double-labeling experiments after the internalization of both transferrin and surface-labeled GLUT4 show that GLUT4 accumulates within peripheral compartments that exclude the transferrin receptor (TfR) at both 15 degrees and 37 degrees C. Thus, GLUT4 is sorted differently from the transferrin receptor as evidenced by the targeting of each protein to distinct early endosomal compartments and by the formation of GSVs. These results suggest that the sorting of GLUT4 from TfR may occur primarily at the level of the plasma membrane into distinct endosomes and that the organization of the endocytic system in CHO cells more closely resembles that of neuroendocrine cells than previously appreciated.     

Targeting of the synaptic vesicle protein synaptobrevin in the axon of cultured hippocampal neurons: evidence for two distinct sorting steps

Synaptic vesicles are concentrated in the distal axon, far from the site of protein synthesis. Integral membrane proteins destined for this organelle must therefore make complex targeting decisions. Short amino acid sequences have been shown to act as targeting signals directing proteins to a variety of intracellular locations. To identify synaptic vesicle targeting sequences and to follow the path that proteins travel en route to the synaptic vesicle, we have used a defective herpes virus amplicon expression system to study the targeting of a synaptobrevin-transferrin receptor (SB-TfR) chimera in cultured hippocampal neurons. Addition of the cytoplasmic domain of synaptobrevin onto human transferrin receptor was sufficient to retarget the transferrin receptor from the dendrites to presynaptic sites in the axon. At the synapse, the SB-TfR chimera did not localize to synaptic vesicles, but was instead found in an organelle with biochemical and functional characteristics of an endosome. The chimera recycled in parallel with synaptic vesicle proteins demonstrating that the nerve terminal efficiently sorts transmembrane proteins into different pathways. The synaptobrevin sequence that controls targeting to the presynaptic endosome was not localized to a single, 10- amino acid region of the molecule, indicating that this targeting signal may be encoded by a more distributed structural conformation. However, the chimera could be shifted to synaptic vesicles by deletion of amino acids 61-70 in synaptobrevin, suggesting that separate signals encode the localization of synaptobrevin to the synapse and to the synaptic vesicle.     

Impaired development of Th2 cells in IL-13-deficient mice

Synaptic vesicles can be coated in vitro in a reaction that is ARF-, ATP-, and temperature-dependent and requires synaptic vesicle membrane proteins. The coat is largely made up of the heterotetrameric complex, adaptor protein 3, recently implicated in Golgi-to-vacuole traffic in yeast. Depletion of AP3 from brain cytosol inhibits small vesicle formation from PC12 endosomes in vitro. Budding from washed membranes can be reconstituted with purified AP3 and recombinant ARF1. We conclude that AP3 coating is involved in at least one pathway of small vesicle formation from endosomes.     

Anatomo-clinical conference: bronchiolo-alveolar cancer and confusion

Rapid membrane recycling in nerve terminals is required to maintain rapid synaptic transmission. Following the fusion of synaptic vesicles with synaptic plasma membranes, recycling can occur via clathrin-coated vesicles (CCVs) [1-3]. The fate of these vesicles is uncertain: they could simply uncoat and acquire other proteins from the cytosol to regenerate synaptic vesicles or they may fuse with endosomal structures from which synaptic vesicles could then bud. We have purified both CCVs and synaptic vesicles from rat brain, and measured the ability of these vesicle fractions to take up the excitatory neurotransmitter glutamic acid. We found that the normalized levels of glutamate uptake by the two types of vesicle were very similar. For each vesicle fraction, uptake required ATP and Cl- and could be fully inhibited by the specific vacuolar proton pump (v-ATPase) inhibitor concanamycin. We suggest that this ability to refill vesicles with neurotransmitter at the earliest intermediate on the recycling pathway - the CCV - may allow uncoated vesicles to immediately enter the releasable pool without sacrificing the quantal nature of neurotransmitter release.     

High-resolution density gradient electrophoresis of subcellular organelles and proteins under nondenaturing conditions

We have developed a density gradient electrophoresis device (DGE) and used it for the preparative separation of various endocytic organelles that are hard to separate by other means. Our separation by DGE of late endosomal vesicles, recycling vesicles, early endosomes and plasma membranes is unmatched. Using the same DGE device, we performed preparative high-resolution rate zonal separation of proteins using amphoteric buffers as originally described by Bier (Electrophoresis 1993, 14, 1011-1018). Isoforms of bovine beta-lactoglobulin, human apo-transferrin, and bovine erythrocyte carbonic anhydrase that have isoelectric points within 0.8 pH units were readily separated even in the absence of nonionic detergents. The DGE apparatus is inexpensive and has unique separation abilities for vesicles and proteins.     

ARF is required for maintenance of yeast Golgi and endosome structure and function

ADP ribosylation factor (ARF) is thought to play a critical role in recruiting coatomer (COPI) to Golgi membranes to drive transport vesicle budding. Yeast strains harboring mutant COPI proteins exhibit defects in retrograde Golgi to endoplasmic reticulum protein transport and striking cargo-selective defects in anterograde endoplasmic reticulum to Golgi protein transport. To determine whether arf mutants exhibit similar phenotypes, the anterograde transport kinetics of multiple cargo proteins were examined in arf mutant cells, and, surprisingly, both COPI-dependent and COPI-independent cargo proteins exhibited comparable defects. Retrograde dilysine-mediated transport also appeared to be inefficient in the arf mutants, and coatomer mutants with no detectable anterograde transport defect exhibited a synthetic growth defect when combined with arf1Delta, supporting a role for ARF in retrograde transport. Remarkably, we found that early and medial Golgi glycosyltransferases localized to abnormally large ring-shaped structures. The endocytic marker FM4-64 also stained similar, but generally larger ring-shaped structures en route from the plasma membrane to the vacuole in arf mutants. Brefeldin A similarly perturbed endosome morphology and also inhibited transport of FM4-64 from endosomal structures to the vacuole. Electron microscopy of arf mutant cells revealed the presence of what appear to be hollow spheres of interconnected membrane tubules which likely correspond to the fluorescent ring structures. Together, these observations indicate that organelle morphology is significantly more affected than transport in the arf mutants, suggesting a fundamental role for ARF in regulating membrane dynamics. Possible mechanisms for producing this dramatic morphological change in intracellular organelles and its relation to the function of ARF in coat assembly are discussed.     

The 46-kDa mannose 6-phosphate receptor contains multiple binding sites for clathrin adaptors

The two known mannose 6-phosphate receptors (MPR46 and MPR300) both mediate the transport of Man-6-P-containing lysosomal proteins to lysosomes. However, the MPRs cannot be detected in lysosomes, instead they recycle between the plasma membrane and endosomes and between endosomes and the trans-Golgi network. Both, endocytosis from the plasma membrane and budding of transport vesicles from the trans-Golgi network involves the interaction of the receptor with the clathrin-coated vesicles-associated protein complexes AP1 and AP2. We have analyzed this interaction between the Golgi-restricted AP1 complex and the plasma membrane-restricted AP2 complex with the MPR46 tail in vitro by using a biosensor. AP1 and AP2 both bind to and dissociate from the MPR46 tail with similar kinetics. Using synthetic peptides corresponding to different MPR receptor tail regions in inhibition and binding studies, a common high affinity binding site for AP1 and AP2 and two separate high affinity binding sites for AP1 and AP2, respectively, were identified.     

Synaptic vesicles retain their identity through the endocytic cycle

After fusion of synaptic vesicles with presynaptic membrane and secretion of the contents of the vesicles into the synaptic cleft (a process known as exocytosis), the vesicular membrane is retrieved by endocytosis (internalization) for re-use. Several issues regarding endocytosis at central synapses are unresolved, including the location of membrane retrieval (relative to the active zone, where exocytosis occurs), the time course of various endocytic steps, and the recycling path taken by newly endocytosed membranes. The classical model of synaptic-vesicle recycling, proposed by analogy to other cellular endocytic pathways, involves retrieval of the membrane, fusion of the membrane with endosome-like compartments and, finally, budding of new synaptic vesicles from endosomes, although the endosomal station may not be obligatory. Here we test the classical model by using the fluorescent membrane dye FM1-43 with quantitative fluorescence microscopy. We find that the amount of dye per vesicle taken up by endocytosis equals the amount of dye a vesicle releases on exocytosis; therefore, we conclude that the internalized vesicles do not, as the classical picture suggests, communicate with intermediate endosome-like compartments during the recycling process.     

GPI anchor attachment is required for Gas1p transport from the endoplasmic reticulum in COP II vesicles

Inositol starvation of auxotrophic yeast interrupts glycolipid biosynthesis and prevents lipid modification of a normally glycosyl phosphatidylinositol (GPI)-linked protein, Gas1p. The unanchored Gas1p precursor undergoes progressive modification in the endoplasmic reticulum (ER), but is not modified by Golgi-specific glycosylation. Starvation-induced defects in anchor assembly and protein processing are rapid, and occur without altered maturation of other proteins. Cells remain competent to manufacture anchor components and to process Gas1p efficiently once inositol is restored. Newly synthesized Gas1p is packaged into vesicles formed in vitro from perforated yeast spheroplasts incubated with either yeast cytosol or the purified Sec proteins (COP II) required for vesicle budding from the ER. In vitro synthesized vesicles produced by inositol-starved membranes do not contain detectable Gas1p. These studies demonstrate that COP II components fulfill the soluble protein requirements for packaging a GPI-anchored protein into ER-derived transport vesicles. However, GPI anchor attachment is required for this packaging to occur.     

Evidence for a symmetrical requirement for Rab5-GTP in in vitro endosome-endosome fusion

Early endosome fusion, which has been extensively characterized using an in vitro reconstitution assay, is Rab5-dependent. To examine the requirement for Rab5 on both fusion partners, we prepared cytosol and endosomes depleted of Rab5. Unlike control cytosol, Rab5-depleted cytosol was only marginally active in the in vitro endosome fusion. However, fusion could be restored by the addition of wild-type Rab5 or Rab5 D136N, a mutant whose nucleotide specificity favors xanthine over guanine. The addition of Rab5 D136N restored fusion only in the presence of XTP. In the absence of XTP or in the presence of XDP, Rab5 D136N failed to restore fusion. When fusion was carried out with endosomal vesicles depleted of Rab GTPases (by preincubation of vesicles with GDP dissociation inhibitor), together with cytosol immunodepleted of Rab5, fusion was virtually absent. We then used immunodepleted cytosol and GDP dissociation inhibitor-treated vesicles to determine whether Rab5 is required by both fusion partners. Using separate sets of endosomal vesicles, we found that priming both sets of Rab5-depleted vesicles with Rab5 Q79L, a GTPase-defective mutant, substantially stimulated endosome fusion. Priming one set of vesicles with Rab5 Q79L and a second set of vesicles with Rab5 S34N failed to activate fusion. When both sets of Rab5-depleted vesicles were primed with Rab5 D136N supplemented with XTP, endosome fusion was stimulated, similar to that observed with Rab5 Q79L. However, when one set of vesicles was preincubated with Rab5 D136N plus XTP and the second set with Rab5 D136N and XDP, no stimulation of fusion was observed. We conclude that Rab5-GTP is required on both fusion partners for docking and fusion of early endosomes. To confirm the fusion of Rab5-GTP-positive vesicles in vivo, we expressed GFP-Rab5 Q79L in fibroblasts and observed fusion of Rab5-positive vesicles. We failed to record fusion of Rab5-positive vesicles with Rab5-negative vesicles. We conclude that Rab5-GTP is required on both sets of endosomes for fusion in vitro and in living cells.     

Separation of early steps in endocytic membrane transport

We describe a simple subcellular fractionation scheme aimed at separating early endosomes from the plasma membrane in view of studying the possible arrival of plasma membrane-bound toxins, proteins or other extracellular ligands in endosomes. Plasma membrane proteins were labeled with the impermeable reagent sulfosuccinimidyl-6-(biotinamido)hexanoate (NHS-LC) biotin at 4 degrees C. In a separate set of cells, early endosomes were labeled by internalization of horseradish peroxidase from the medium for 5 min. The first step of the purification, which consists of a step sucrose gradient, led to three fractions, respectively: enriched in biosynthetic membranes (interface 3), in plasma membrane and early endosomes (interface 2), and in late endosomes (interface 1). The second step, in which interface 2 was loaded at the bottom of a 17% Percoll gradient, led to the separation of the plasma membrane, including caveolae and cholesterol-glycolipid rafts, from early endosomes. Western blot analysis of the fractions from the Percoll gradient showed that the transferrin receptor, the small GTPases rab5 and Arf6, as well as annexin II were present both at the plasma membrane and in early endosomes, whereas the caveolar marker caveolin, 1co, migrated only with the biotinylated plasma membrane proteins. We used this fractionation procedure to show that the pore-forming toxin aerolysin does not reach the endocytic compartments of baby hamster kidney (BHK) cells. The procedure should be generally useful in rapidly determining whether extracellular proteins or ligands reach endosomes.     

A helical coat protein recognition domain of the bacteriophage P22 scaffolding protein

The transcytotic pathway followed by the polymeric IgA receptor (pIgR) carrying its bound ligand (dIgA) from the basolateral to the apical surface of polarized MDCK cells has been mapped using morphological tracers. At 20 degreesC dIgA-pIgR internalize to interconnected groups of vacuoles and tubules that comprise the endosomal compartment and in which they codistribute with internalized transferrin receptors (TR) and epidermal growth factor receptors (EGFR). Upon transfer to 37 degreesC the endosome vacuoles develop long tubules that give rise to a distinctive population of 100-nm-diam cup-shaped vesicles containing pIgR. At the same time, the endosome gives rise to multivesicular endosomes (MVB) enriched in EGFR and to 60-nm-diam basolateral vesicles. The cup-shaped vesicles carry the dIgA/pIgR complexes to the apical surface where they exocytose. Using video microscopy and correlative electron microscopy to study cells grown thin and flat we show that endosome vacuoles tubulate in response to dIgA/pIgR but that the tubules contain TR as well as pIgR. However, we show that TR are removed from these dIgA-induced tubules via clathrin-coated buds and, as a result, the cup-shaped vesicles to which the tubules give rise become enriched in dIgA/pIgR. Taken together with the published information available on pIgR trafficking signals, our observations suggest that the steady-state concentrations of TR and unoccupied pIgR on the basolateral surface of polarized MDCK cells are maintained by a signal-dependent, clathrin-based sorting mechanism that operates along the length of the transcytotic pathway. We propose that the differential sorting of occupied receptors within the MDCK endosome is achieved by this clathrin-based mechanism continuously retrieving receptors like TR from the pathways that deliver pIgR to the apical surface and EGFR to the lysosome.     

Morphology of the yeast endocytic pathway

Positively charged Nanogold (Nanoprobes, Stony Brook, NY) has been developed as a new marker to follow the endocytic pathway in yeast. Positively charged Nanogold binds extensively to the surface of yeast spheroplasts and is internalized in an energy-dependent manner. Internalization of gold is blocked in the end3 mutant. During a time course of incubation of yeast spheroplasts with positively charged Nanogold at 15 degrees C, the gold was detected sequentially in small vesicles, a peripheral, vesicular/tubular compartment that we designate as an early endosome, a multivesicular body corresponding to the late endosome near the vacuole, and in the vacuole. Experiments examining endocytosis in the sec18 mutant showed an accumulation of positively charged Nanogold in approximately 30-50 nm diameter vesicles. These vesicles most likely represent the primary endocytic vesicles as no other intermediates were detected in the mutant cells, and they correspond in size to the first vesicles detected in wild-type spheroplasts at 15 degrees C. These data lend strong support to the idea that the internalization step of endocytosis in yeast involves formation of small vesicles of uniform size from the plasma membrane.     

Association of Rab25 and Rab11a with the apical recycling system of polarized Madin-Darby canine kidney cells

Recent evidence suggests that apical and basolateral endocytic pathways in epithelia converge in an apically located, pericentriolar endosomal compartment termed the apical recycling endosome. In this compartment, apically and basolaterally internalized membrane constituents are thought to be sorted for recycling back to their site of origin or for transcytosis to the opposite plasma membrane domain. We report here that in the epithelial cell line Madin-Darby Canine Kidney (MDCK), antibodies to Rab11a label an apical pericentriolar endosomal compartment that is dependent on intact microtubules for its integrity. Furthermore, this compartment is accessible to a membrane-bound marker (dimeric immunoglobulin A [IgA]) internalized from either the apical or basolateral pole, functionally defining it as the apical recycling endosome. We have also examined the role of a closely related epithelial-specific Rab, Rab25, in the regulation of membrane recycling and transcytosis in MDCK cells. When cDNA encoding Rab25 was transfected into MDCK cells, the protein colocalized with Rab11a in subapical vesicles. Rab25 transfection also altered the distribution of Rab11a, causing the coalescence of immunoreactivity into multiple denser vesicular structures not associated with the centrosome. Nevertheless, nocodazole still dispersed these vesicles, and dimeric IgA internalized from either the apical or basolateral membrane was detected in endosomes labeled with antibodies to both Rab11a and Rab25. Overexpression of Rab25 decreased the rate of IgA transcytosis and of apical, but not basolateral, recycling of internalized ligand. Conversely, expression of the dominant-negative Rab25T26N did not alter either apical recycling or transcytosis. These results indicate that both Rab11a and Rab25 associate with the apical recycling system of epithelial cells and suggest that Rab25 may selectively regulate the apical recycling and/or transcytotic pathways.     

In vitro binding of synaptic vesicles to the synaptic plasma membrane: lack of effect of beta-bungarotoxin

To help characterize the mechanisms of neurotransmitter release, and the role of the specific neurotoxin beta-bungarotoxin in inhibiting release, the interaction of synaptic vesicles with the synaptic plasma membrane was investigated using two in vitro systems. Binding of radiolabeled synaptic vesicles to immobilized synaptic plasma membrane was specific, protein-dependent, and modulated by phosphorylation of membrane proteins. Stimulation of phosphorylation by phorbol ester increased binding, and reduction of phosphorylation by alkaline phosphatase or staurosporine reduced binding. beta-Bungarotoxin did not alter basal binding of synaptic vesicles to synaptic plasma membrane, nor did it affect the increase in binding induced by phorbol esters. Under conditions which stimulate acetylcholine release from synaptosomes, both phorbol ester and 4-aminopyridine caused an increase in attachment of the synaptic vesicle marker protein synaptophysin to the synaptic plasma membrane. beta-Bungarotoxin did not alter the change in localization of synaptophysin induced by either drug, under conditions in which it inhibits ACh release induced by 4-aminopyridine. It is concluded that beta-bungarotoxin inhibition probably does not occur at the level of the interaction of the synaptic vesicle and the synaptic plasma membrane, but occurs at an earlier stage in the neurotransmission process.     

ATP- and cytosol-dependent release of adaptor proteins from clathrin-coated vesicles: A dual role for Hsc70

Clathrin-coated vesicles (CCV) mediate protein sorting and vesicular trafficking from the plasma membrane and the trans-Golgi network. Before delivery of the vesicle contents to the target organelles, the coat components, clathrin and adaptor protein complexes (APs), must be released. Previous work has established that hsc70/the uncoating ATPase mediates clathrin release in vitro without the release of APs. AP release has not been reconstituted in vitro, and nothing is known about the requirements for this reaction. We report a novel quantitative assay for the ATP- and cytosol- dependent release of APs from CCV. As expected, hsc70 is not sufficient for AP release; however, immunodepletion and reconstitution experiments establish that it is necessary. Interestingly, complete clathrin release is not a prerequisite for AP release, suggesting that hsc70 plays a dual role in recycling the constituents of the clathrin coat. This assay provides a functional basis for identification of the additional cytosolic factor(s) required for AP release.     

Biochemical evidence that Semliki Forest virus obtains its envelope from the plasma membrane of the host cell

The data from chemical studies and electron microscopy suggest that Semliki Forest virus obtains its envelope by budding into the medium from the plasma membrane of the host cell. Biochemical evidence for this phenomenon, however, has not been published. Therefore, we undertook a series of pulse-chase studies so that we might quantitatively evaluate the importance of the budding mechanism in the morphogenesis of Semliki Forest virus. Baby hamster kidney cells (clone 13) were grown in culture and infected with Semliki Forest virus. The cells were exposed to [4,5-3H] leucine for 20 min and the subsequent incorporation of the label into virus proteins associated with cytoplasmic membranes and extracellular virus was determined. Initial experiments were conducted with microsomes and a precursor-product relationship was demonstrated between viral proteins in the microsomes and in extracellular virus. Further studies were performed with endoplasmic reticulum and plasma membrane preparations. Maximal incorporation of [3H] leucine was observed in the viral proteins located in the endoplasmic reticulum at the end of a 20-min pulse period; greater than 50% of this activity had disappeared within 2 h. The plasma membrane fraction contained no radioactivity at the end of the pulse period; subsequently, maximal labeling of the viral proteins in the plasma membrane occurred 4 h into the chase period and these labeled proteins had disappeared from this membrane 11 h after the pulse. At this time maximal incorporation of the labeled proteins into extracellular virus was observed. These data are consistent with a precursor-product relationship between the viral proteins in the endoplasmic reticulum which migrate to the plasma membrane and are subsequently incorporated into extracellular virus. All the radioactivity in the extracellular virus appears to have been derived from viral proteins associated with the plasma membrane of the cell. Therefore, mechanisms for the morphogenesis of Semliki Forest virus (in baby hamster kidney cells), other than budding from the plasma membrane, are unlikely to be of quantitative importance.     

Syntaxin 13 mediates cycling of plasma membrane proteins via tubulovesicular recycling endosomes

Endocytosis-mediated recycling of plasma membrane is a critical vesicle trafficking step important in diverse biological processes. The membrane trafficking decisions and sorting events take place in a series of heterogeneous and highly dynamic organelles, the endosomes. Syntaxin 13, a recently discovered member of the syntaxin family, has been suggested to play a role in mediating endosomal trafficking. To better understand the function of syntaxin 13 we examined its intracellular distribution in nonpolarized cells. By confocal immunofluorescence and electron microscopy, syntaxin 13 is primarily found in tubular early and recycling endosomes, where it colocalizes with transferrin receptor. Additional labeling is also present in endosomal vacuoles, where it is often found in clathrin-coated membrane areas. Furthermore, anti-syntaxin 13 antibody inhibits transferrin receptor recycling in permeabilized PC12 cells. Immunoprecipitation of syntaxin 13 revealed that, in Triton X-100 extracts, syntaxin 13 is present in a complex(es) comprised of betaSNAP, VAMP 2/3, and SNAP-25. This complex(es) binds exogenously added alphaSNAP and NSF and dissociates in the presence of ATP, but not ATPgammaS. These results support a role for syntaxin 13 in membrane fusion events during the recycling of plasma membrane proteins.