©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Appendage Domain of -Adaptin Is a High Affinity Binding Site for Dynamin (*)

Li-Hsien Wang (1), Thomas C. Südhof (2) (3), Richard G. W. Anderson (1)(§)

From the (1) Departments of Cell Biology and Neuroscience and (2) Molecular Genetics and the (3) Howard Hughes Medical Institute, the University of Texas Southwestern Medical Center, Dallas, Texas 75235

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Dynamin is a GTPase that appears to be required for endocytosis. Even though this molecule is known to be in surface-coated pits, the identity of the resident coat proteins that account for this localization is not known. Here we show that dynamin is one of three synaptic terminal proteins that bind with specificity to the appendage domain of -adaptin. Binding is sensitive to both salt and pH levels but is not affected by nucleotides. Using recombinant dynamin expressed in SF9 cells, we estimate that the binding affinity is 200 nM. Binding does not require GTP, and the GTPase activity of dynamin is not stimulated by this interaction. These results suggest that the COOH terminus of -adaptin may be a domain within AP2 that mediates the initial interactions between dynamin and surface-coated pits. This may be an essential step in the regulation of coated pit budding.


INTRODUCTION

Clathrin AP2 (adaptor) together with triskelion are the major structural subunits of plasma membrane clathrin-coated pits (1, 2) . AP2 is a hetero-oligomer composed of two 100-kDa adaptin molecules ( and ) plus one copy each of a 50- and a 17-kDa protein (1) . AP2 is only found in plasma membrane-coated pits, but a similarly constructed subunit, called AP1, serves the same purpose in coated pits of the Golgi apparatus (1) . One of the chief differences between the two subunits is that the - and -adaptins are replaced in the AP1 subunit with - and `-adaptins. A major function of both AP subunits is to provide a linkage between the triskelion lattice and the membranes of coated pits and vesicles (3, 4) .

Rapid freeze, deep etch images of the AP subunit show that it consists of a brick-shaped core with two appendages that extend from either side (5) . The appendages correspond to the 300-amino acid COOH terminus of each adaptin molecule. The core, by contrast, is constructed from the remainder of the two adaptins (designated the trunk domain (1) ) plus the other two protein components. The architecture of this subunit, together with the sequence variability of the COOH terminus of the adaptin molecule (6) , suggests that the appendage domain may interact with molecules that transiently associate with clathrin-coated pits during endocytosis (2) . So far there have not been any molecules identified that bind to this domain. The trunk domain, on the other hand, has been shown to interact with the cytoplasmic tail of several receptors that cluster in coated pits (7, 8, 9) . In addition, this domain is known to bind tightly to synaptotagmin I (4, 10) and to clathrin (4, 11) .

A molecule that has emerged as an important functional component of the clathrin-coated pit is dynamin (12, 13) . The GTPase activity of this molecule appears to be required for the conversion of a plasma membrane-coated pit into a coated vesicle (14, 15) . Furthermore, dynamin has recently been found to bind tightly to several different signal transducing molecules that contain SH3 domains (16, 17, 18) . The protein responsible for directing dynamin to coated pits is not known, but a likely candidate is one of the adaptin molecules (15) . We constructed fusion proteins between glutathione S-transferase (GST)() and three different domains within the - and -adaptins to see if any other of these proteins would interact with dynamin as well as other cytosolic proteins in brain extracts. We found that dynamin binds with specificity and high affinity to the appendage domain of -adaptin.


EXPERIMENTAL PROCEDURES

Materials

The following reagents were obtained from Sigma: ampicillin (A-9518), benzamidine (B-6506), HEPES (H-3375), EDTA (ED2SS), glutathione (G-4251), glutathione-agarose (G-4510), magnesium chloride ), potassium chloride (P-3911), pepstatin A (P-4265), sodium chloride (S-9625), Tween 20 (P-1379), Triton X-100 (X-100), trichloroacetic acid (T-6399), monoclonal anti--adaptin (clone 100/3, A-4200), and monoclonal anti-assembly protein AP180 (clone AP180-I, A-4825). Phenylmethanesulfonyl fluoride was purchased from Boehringer Mannheim. 1,4-Dithio-DL-threitol was from Fluka Chemical Corp. (Ronkonkomam, NY). Leupeptin was obtained from Peptide Institute Inc. (Osaka, Japan). Isopropyl--D-thiogalactopyranoside was purchased from Stratagene (La Jolla, CA). Horseradish peroxidase-conjugated goat IgG fractions against mouse IgG ( (50) or goat IgG fractions against rabbit IgG (55676) were from Organon Teknika Corp. (Durham, NC). The ECL detection kit was purchased from Amersham Corp. Prokaryotic expression vector pGEX2T was obtained from Pharmacia Biotech Inc. The TA cloning kit was from Invitrogen Corp. (San Diego, CA). Reagents used for bacterial culture were purchased from Difco. All restriction enzymes were obtained from Promega Corp. (Madison, WI). The reagents and apparatus for acrylamide gel electrophoresis were purchased from Bio-Rad. The Immobilon-P blotting membrane was obtained from Millipore Corp. (Bedford, MA). The agarose and the equipment for agarose gel electrophoresis were from Life Technologies, Inc. Monoclonal antibodies that recognize -adaptin (AC1M11) and full-length mouse cDNAs of - and -adaptin were kindly provided by Dr. Margaret S. Robinson from the University of Cambridge, UK. Monoclonal IgG against clathrin (OZ-71) and polyclonal antisera against dynamin were prepared as previously described (19, 32) .

Buffers

The cytosol buffer contained 20 mM HEPES, pH 7.2, 100 mM KCl, 2 mM MgCl, and 1 mM dithiothreitol. The protease inhibitor mixture was 10 µM leupeptin, 1 µg/ml pepstatin A, 0.5 mM benzamidine, and 1 mM phenylmethanesulfonyl fluoride in cytosol buffer. Tris-buffered saline included 20 mM Tris and 137 mM NaCl, pH 7.6. The antibody buffer contained Tris-buffered saline plus 1% dry milk and 0.1% Tween 20.

Preparation of Fusion Proteins

The construction of GST fusion proteins containing different adaptin domains was performed according to standard methods. The appropriate regions of cDNA for each fusion protein were amplified from plasmids containing a full-length cDNA insert of - or -adaptin by using polymerase chain reaction. The sequences of the first seven and the last seven codons of each segment were synthesized as the primers. The names and inserted regions of the three fusion proteins used in this experiment are: GSTT, amino acids 1-620 of -adaptin; GSTA, amino acids 701-938 of -adaptin; and GSTA, amino acids 704-822 of -adaptin. Amplified fragments were subcloned into the pCR II cloning vector, and the EcoRI fragments were subsequently transferred into the pGEX2T expression vector. The plasmids containing the cDNAs encoding for all fusion proteins were then transformed into either BL21 or HB101 Escherichia coli strains. The fusion proteins were induced and attached to the affinity matrix as described below. A 10-ml aliquot of the overnight culture was inoculated into 1 liter of fresh LB medium containing 80 µg/ml ampicillin and grown at 37 °C with vigorous shaking. When the density of the culture reached about A0.6, the flasks were transferred to room temperature, and the production of fusion proteins was induced by adding isopropyl--D-thiogalactopyranoside to a final concentration of 50 µM for another 4 h with shaking. The bacteria were harvested by centrifuging at 4000 g for 10 min at 4 °C, and the resulting pellet was resuspended in 40 ml of ice-cold cytosol buffer. The suspension was then sonicated for four 15-s pulses with a half-inch probe and with the power setting at 3 (Ultrasonic Processor model W-380, Heat System-Ultrasonics Inc.). The debris was removed by spinning the sonicated lysate at 12,000 g for 20 min at 4 °C. The supernatant was carefully transferred to a new tube and supplemented with Triton X-100 to a final concentration of 1%. The resulting supernatant was incubated with 1.5 ml of preswollen glutathione-agarose beads for at least 2 h at 4 °C with tumbling. The beads were then washed three times by repeated centrifugation and resuspension with 40 ml of cytosol buffer containing 0.1% Triton X-100. After the third wash, the beads with attached fusion proteins were stored in the form of 50% slurry at 4 °C. The fusion proteins remained stable without any degradation for at least 1 month.

Preparation of Cellular Extract

Bovine brains were obtained from a local slaughterhouse. After removing meninges, whole brain tissue was dissected into small pieces and snap-frozen in liquid nitrogen. All the following procedures were performed at 4 °C. Bovine brain membrane extract was prepared by homogenizing frozen brain tissue in cytosol buffer containing the protease inhibitor mixture at a ratio of 1 ml of buffer to 1 g (wet weight) of tissue. The mixture was centrifuged for 1 h at 100,000 g at 4 °C, and the pellet was resuspended in the same volume of fresh cytosol buffer. Triton X-100 was added slowly with stirring to a final concentration of 1%, and then the mixture was incubated at 4 °C for 1 h. The mixture was centrifuged at 100,000 g for 1 h at 4 °C, and the supernatant fraction was collected (designated brain extract). The protein concentration of this extract was approximately 15 mg/ml.

Identification of AP Binding Proteins

The membrane extract was precleared by incubating each fraction of brain extract with 75 µl of packed glutathione-agarose beads for 4 h at 4 °C. The beads were removed by centrifugation, and 75 µl of beads containing the indicated fusion proteins was added. The mixture was then incubated overnight at 4 °C with tumbling. The glutathione-agarose beads were recovered by spinning at 8500 g for 2 min at 4 °C. The beads were washed 6 times by repeated resuspension and centrifugation with 1 ml of cytosol buffer containing 0.1% Triton X-100. Bound proteins were eluted by incubating beads in 650 µl of 50 mM glutathione in cytosol buffer for 10 min and spinning at 8500 g to remove the beads. The supernatant fraction was precipitated with trichloroacetic acid, and one-fifth of each sample was analyzed by electrophoresis on a 10% SDS gel. After separation, proteins were visualized by Coomassie Blue R-250 staining.

Electrophoresis and Immunoblots

Protein samples were separated by SDS-polyacrylamide gel electrophoresis and then transferred to Immobilon-P polyvinylidene difluoride membranes. After transferring, the blots were preblocked for 60 min with 5% dry milk (Carnation) in Tris-buffered saline containing 0.5% Tween 20. Both primary antibody and secondary antibody were diluted in antibody buffer. Anti-dynamin rabbit polyclonal antiserum (E765) was diluted at a ratio of 1/2,000. AC1M11, AP180-1, and 100/3 ascites fluids were used at dilutions of 1/2,000, 1/15,000, and 1/200, respectively. OZ71-purified IgG was used at a concentration of 20 µg/ml. The preblocked membranes were rinsed twice with antibody buffer and incubated with primary antibody solutions for 1-2 h at room temperature. The appropriate second antibody coupled to horseradish peroxidase was used at a dilution of 1/30,000 and detected by enhanced chemiluminescence reagents. The relative amount of dynamin was determined by densitometry with a personal densitometer (Molecular Dynamics).

Peptide Microsequencing

Proteins eluted from the GSTA fusion proteins were transferred onto polyvinylidene difluoride membranes. The membranes were stained with Ponceau S. The bands were then excised and digested in situ with Lyc-C endoprotease. The resulting peptides were separated by reverse phase high pressure liquid chromatography and sequenced using an automated protein sequenator.

Other Methods

Protein concentration was determined with Bio-Rad Bradford protein assay reagent using bovine serum albumin as the standard.


RESULTS

To see if -adaptin may be involved in targeting dynamin to surface-coated pits, we constructed plasmids that express either the -adaptin appendage (GSTA) and trunk (GSTT) domains or the -adaptin appendage (GSTA) domain fused to glutathione S-transferase. Either GST alone or each of the fusion proteins was then attached to glutathione beads. Brain is a rich source of dynamin (20) , so we incubated the beads overnight at 4 °C in the presence or absence of 15 mg/ml Triton X-100 bovine brain extract (Fig. 1 A). The beads were washed with 50 mM glutathione, and the eluted proteins were separated by gel electrophoresis before staining with Coomassie Blue. Each bead contained approximately the same amount of fusion protein (Fig. 1 A, heavy band in each lane). GST alone did not bind any proteins (Fig. 1 A, lanes 1 and 2). GSTA specifically bound four proteins with molecular weights of 180,000, 120,000, 100,000, and 94,000 (Fig. 1 A, lane 4). GSTA, by contrast, bound specifically a 180-kDa protein plus a cluster of proteins that migrated at about 100 kDa (Fig. 1 A, lane 6). GSTT did not bind any proteins in this molecular weight range. Therefore, the plasma membrane- and Golgi-specific appendage domains appeared to form a complex with different sets of proteins present in the brain extract.


Figure 1: Identification of proteins that bind selectively to the appendage domain of - and -adaptin. The indicated GST fusion protein was attached to glutathione-agarose beads, incubated in the presence (+) or absence () of a Triton X-100 brain extract (15 mg/ml) and then eluted with 50 mM glutathione. A, Coomassie Blue staining of the eluted fractions from each of the indicated beads. B, sequence analysis of four prominent bands that bound to the GSTA. We compared the bovine sequences obtained with those sequences in data base and found perfect matches between the 180-kDa band and mouse AP180 ( top sequence, residues 135-149), the 120-kDa band and chicken amphiphysin ( middle sequence, residues 31-47), and the 100-kDa band and rat dynamin ( bottom sequence, residues 90-107).



We used microsequencing to identify the four proteins that were in the GSTA complex (Fig. 1 B). Each of the bands was cut from the gel and sequenced by standard methods. We obtained multiple sequences () from each protein (legend to Fig. 1 B) and identified the 180-kDa band as AP180 (21, 22) , the 120-kDa band as amphiphysin (23) , and the 100-kDa band as dynamin (20) . One of the sequences obtained from the 94-kDa band matched a sequence in a yeast protein of unknown function (GenBankaccession number U14913).

We used immunoblotting to further characterize the various proteins that bound to GSTA, GSTT, and GSTA (Fig. 2). Anti-dynamin IgG reacted with the 100-kDa band in the GSTA complex (Fig. 2, lane 2). This antibody did not blot any of the proteins that bound to either GSTT or GSTA (Fig. 2, lanes 3 and 4). When we blotted with an anti-AP180 IgG, we found a strongly reactive band in the GSTA complex (Fig. 2, lane 14) but very little reactivity in the GSTA complex (Fig. 2, lane 16). Because a protein of similar molecular weight was associated with GSTA (Fig. 1 A, lane 8), we immunoblotted each lane with anti-clathrin IgG. This antibody recognized the 180-kDa band in the GSTA complex (Fig. 2, lane 20). Anti-clathrin IgG did not bind to any of the proteins in the GSTA complex (Fig. 2, lane 18).


Figure 2: Immunoblot identification of proteins that bind to appendage domain of adaptin fusion proteins. The indicated fusion protein was incubated in the presence of brain membrane extract and washed with glutathione, and the eluted proteins were separated by gel electrophoresis. The indicated antibodies were then used to immunoblot each of the fractions.



The presence of clathrin in the GSTA complex suggested that the cluster of associated proteins in the 100,000 molecular weight region (Fig. 1, lane 8) might correspond to the adaptins. Therefore, we blotted each of the four fusion protein complexes with either anti--adaptin IgG or anti--adaptin IgG. Surprisingly, both antibodies recognized the bands associated with GSTA (Fig. 2, lanes 8 and 12). There were no immunoreactive bands in either the GSTA or the GSTT lane (Fig. 2, lanes 6, 7, 10, and 11). The differential intensity of the bands suggested that the GSTA complex bound more -adaptin than -adaptin. These results indicate that the -appendage domain selectively interacts with APs but is not able to discriminate between AP2 and AP1.

The expression of GTPase-deficient dynamin in fibroblasts has a dominant negative affect on coated pit-mediated endocytosis (14, 15) . Therefore, we analyzed how nucleotides and ionic conditions affected the association of dynamin with GSTA. The addition of GTP, GTPS, AMP-PNP, ATPS, or ATP to the brain extract had no effect on the binding of either dynamin or the other proteins in the complex (Fig. 3 A, upper and lower panels). We also found that GTP did not stimulate the dissociation of dynamin that had been prebound to GSTA (data not shown). On the other hand, when the salt concentration of the extract was increased, there was a reciprocal decline in the amount of dynamin that bound to the beads (Fig. 3 B). Above 400 mM KCl, dynamin did not bind at all (Fig. 3 B, lower panel). The binding of the other three proteins, however, was relatively insensitive to salt concentration (Fig. 3 B, upper panel). We also found that dynamin binding was extremely sensitive to pH levels (Fig. 3 C). Good binding was observed at neutral pH but rapidly declined as the pH was lowered (Fig. 3 C, lower panel). At pH 6.0, there was more than a 90% decrease in the amount of bound dynamin. pH levels had little effect on the binding of the other proteins in the complex (Fig. 3 C, upper panel). Both pH and salt were also found to dissociate dynamin that had been prebound to GSTA (data not shown).


Figure 3: Effects of nucleotides ( A), ionic strength ( B), and pH ( C) on the binding of dynamin to the -adaptin appendage domain. GSTA was attached to glutathione-agarose beads and incubated with brain membrane extract that had the indicated nucleotide ( A), KCl concentration ( B), or pH ( C) as labeled at the top of each lane in the upper panels. The bound proteins were eluted with glutathione and analyzed by electrophoresis. The gels were either stained by Coomassie Blue ( upper panel) or transferred and immunoblotted with dynamin-specific antiserum ( lower panel). The relative amount of bound dynamin was quantified by densitometry ( lower panel).



As a final test of binding specificity, we measured the affinity of interaction between dynamin and the GSTA. The fusion protein was attached to beads and incubated in the presence of different concentrations of brain extract (Fig. 4 A). As the amount of extract increased, there was an increase in the amount of dynamin that bound. Binding began to saturate at an extract concentration of 12-15 mg/ml. We estimate from this curve that half-maximal binding occurred at 5 mg/ml extract. Because 0.4% of the brain protein is dynamin (24) , this corresponds to a dynamin concentration of 2 10 M.


Figure 4: High affinity interaction of dynamin with the appendage domain. GSTA was immobilized on beads and incubated in the presence of the indicated concentration of either brain extract ( A) or recombinant rat dynamin I ( B). Bound proteins were eluted from the beads, separated by gel electrophoresis, and immunoblotted with anti-dynamin antiserum. The dynamin band in each immunoblot was then quantified.



Dynamin could be binding directly to the appendage or be linked to the domain by one or more of the other three proteins present in the GSTA complex. To distinguish between these possibilities, we expressed rat brain dynamin I fused to polyhistidine in SF9 cells and purified the protein. We then incubated GSTA in the presence of various concentrations of the fusion protein and assayed for binding. Fig. 4 B shows that the binding of dynamin I was concentration-dependent and that half-maximal binding occurred at a concentration of 2 10 M (Fig. 4 B). Polyhistidine-tagged dynamin did not bind to GSTA (data not shown). Therefore, the affinity of interaction between GSTA and either the pure protein or the cytosolic dynamin is similar. This suggests that neither a cofactor nor a covalent modification is required for the direct binding of dynamin to the -appendage of AP2.


DISCUSSION

Coated pit budding is arrested in the Drosophila shibire fly without there being any detectable change in the budding of vesicles from the Golgi apparatus (25, 26) . Therefore, only surface-coated pits appear to require this protein to function. Damke et al. (27) have shown that dynamin is located in plasma membrane-coated pits but not coated pits in the Golgi apparatus. The current results offer an explanation for this selectivity. Dynamin bound directly to the -appendage and did not require the presence of the other proteins in the GSTA complex for binding. Other parts of the -adaptin molecule did not interact nor did the -adaptin appendage domain. Although other combinations of coated pit proteins need to be tested to be sure, the high affinity and specificity of binding strongly suggest that -adaptin is responsible for targeting dynamin to surface-coated pits.

Several laboratories have found that dynamin contains an SH3 binding domain capable of interacting with phospholipase C, GRB-2, and phosphatidylinositol 3`-kinase (16, 17, 18) . There is also evidence that amphiphysin, one of the other proteins in the GSTA complex, is an SH3-containing protein that binds dynamin (28) . The presence of dynamin in coated pits places it in a key location to interact with the receptors that bind these SH3-containing, signal-transducing proteins (29) . In this location, dynamin could function as an exchange factor that removes SH3-containing molecules from the cytoplasmic tails of receptors being internalized by coated pits. Therefore, dynamin may be essential for turning off signal transduction as the receptor-hormone complex moves to the lysosome for degradation.

These results do not explain how GTPase-defective dynamin prevents endocytosis, because GTP was not required for dynamin binding to GSTA. The GTPase activity of the dynamin must be required for other steps in the budding reaction. Most likely -adaptin functions as a tethering device that holds dynamin in the proper location to optimize its interaction with other molecules required to complete the endocytosis cascade. We do not know the exact orientation of the AP2 subunit in the coated pit (30) , but the appendage domain could be quite close to the membrane. This would place dynamin in an optimal position to interact with membrane lipids and proteins that might be passing by.

In contrast to dynamin, the other two proteins that were identified in the GSTA complex appear to be brain-specific. Because they are abundant in synapses, they must play a specific role in the endocytosis of synaptic vesicle membrane. A special feature of endocytosis at the synapse is the requirement for extremely rapid membrane retrieval (31) . Therefore, amphiphysin and AP180 may be molecules that accelerate the budding of coated pits at these sites.

  
Table: Sequence and identity of the four proteins that bind to the -appendage domain of AP2

The third sequence from the 94-kDa protein matched a sequence in the GenBankdata base with accession number U14913. This protein has no known function.



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX 75235.

The abbreviations used are: GST, glutathione S-transferase; GTPS, guanosine 5`-3- O-(thio)triphosphate; AMP-PNP, 5`-adenylyl ,-imidodiphosphate; ATPS, adenosine 5`- O-(thiotriphosphate).


ACKNOWLEDGEMENTS

We thank Dr. H.-C. Lin for assistance with the expression and purification of rat brain dynamin 1a, Dr. Clive Slaughter for doing the sequencing, and Dr. M. S. Robinson for providing monoclonal antibodies against -adaptin and the full-length cDNA for - and -adaptin.


REFERENCES
  1. Keen, J. H. (1990) Annu. Rev. Biochem. 59, 415-438 [CrossRef][Medline] [Order article via Infotrieve]
  2. Pearse, B. M. F., and Robinson, M. S. (1990) Annu. Rev. Cell Biol. 6, 151-171 [CrossRef]
  3. Crowther, R. A., and Pearse, B. M. F. (1981) J. Cell Biol. 91, 790-797 [Abstract/Free Full Text]
  4. Peeler, J. S., Donzell, W. C., and Anderson, R. (1993) J. Cell Biol. 120, 47-54 [Abstract]
  5. Heuser, J. E., and Keen, J. H. (1988) J. Cell Biol. 107, 877-886 [Abstract]
  6. Kirchhausen, T., Nathanson, K. L., Matsui, W., Vaisberg, A., Chow, E. P., Burne, C., Keen, J. H., and Davis, A. E. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 2612-2616 [Abstract]
  7. Beltzer, J. P., and Spiess, M. (1991) Eur. Mol. Biol. Org. J. 10, 3735-3742 [Abstract]
  8. Sosa, M. A., Schmidt, B., Von Figura, K., and Hille-Rehfeld, A. (1993) J. Biol. Chem. 268, 12537-12543 [Abstract/Free Full Text]
  9. Sorkin, A., and Carpenter, G. (1993) Science 261, 612-615 [Medline] [Order article via Infotrieve]
  10. Zhang, J. Z., Davletov, B. A., Südhof, T. C., and Anderson, R. G. W. (1994) Cell 78, 751-760 [Medline] [Order article via Infotrieve]
  11. Mahaffey, D. T., Peeler, J. S., Brodsky, F. M., and Anderson, R. G. W. (1990) J. Biol. Chem. 265, 16514-16520 [Abstract/Free Full Text]
  12. Van der Bliek, A. M., and Meyerowitz, E. M. (1991) Nature 351, 411-414 [CrossRef][Medline] [Order article via Infotrieve]
  13. Chen, M. S., Obar, R. A., Schroeder, C. C., Austin, T. W., Poodry, C. A., Wadsworth, S. C., and Vallee, R. B. (1991) Nature 351, 583-586 [CrossRef][Medline] [Order article via Infotrieve]
  14. Van der Bliek, A. M., Redelmeier, T. E., Damke, H., Tisdale, E. J., Meyerowitz, E. M., and Schmid, S. L. (1993) J. Cell Biol. 122 , 553-563 [Abstract]
  15. Herskovits, J. S., Burgess, C. C., Obar, R. A., and Vallee, R. B. (1993) J. Cell Biol. 122, 565-578 [Abstract]
  16. Booker, G. W., Gout, I., Downing, A. K., Driscoll, P. C., Boyd, J., Waterfield, M. D., and Campbell, I. D. (1993) Cell 73, 813-822 [Medline] [Order article via Infotrieve]
  17. Gout, I., Dhand, R., Hiles, I. D., Fry, M. J., Panayotou, G., Das, P., Truong, O., Totty, N. F., Hsuan, J., Booker, G. W., Campbell, I. D., and Waterfield M. D. (1993) Cell 75, 25-36 [Medline] [Order article via Infotrieve]
  18. Seedorf, K., Kostka, G., Lammers, R., Bashkin, P., Daly, R., Burgess, W. H., van der Bliek, A. M., Schlessinger, J., and Ullrich, A. (1994) J. Biol. Chem. 269, 16009-16014 [Abstract/Free Full Text]
  19. Pathak, R. K., and Anderson, R. G. W. (1989) J. Histochem. Cytochem. 37, 69-74 [Abstract]
  20. Obar, R. A., Collins, C. A., Hammarback, J. A., Shpetner, H. S., and Vallee, R. B. (1990) Nature 347, 256-261 [CrossRef][Medline] [Order article via Infotrieve]
  21. Morris, S. A., Schröder, S., Plessmann, U., Weber, K., and Ungewickell, E. (1993) Eur. Mol. Biol. Org. J. 12(2) , 667-675
  22. Zhou, S., Tannery, N. H., Yang, J., Puszkin, S., and Lafer, E. M. (1993) J. Biol. Chem. 268, 12655-12662 [Abstract/Free Full Text]
  23. Lichte, B., Veh, R. W., Meyer, H. E., and Kilimann, M. W. (1992) Eur. Mol. Biol. Org. J. 11, 2521-2530 [Abstract]
  24. Liu, J. P., Powell, K. A., Südhof, T. C., and Robinson, P. J. (1994) J. Biol. Chem. 269, 21043-21050 [Abstract/Free Full Text]
  25. Poodry, C. A., Hall, L., and Suzuki, D. T. (1973) Dev. Biol. 32, 373-386 [Medline] [Order article via Infotrieve]
  26. Poodry, C. A., and Edgar, L. (1979) J. Cell Biol. 81, 520-527 [Abstract]
  27. Damke, H., Baba, T., Warnock, D. E., and Schmid, S. L. (1994) J. Cell Biol. 127, 915-934 [Abstract]
  28. David, C., McPherson, P. S., Cho, Y., Solimena, M., and De Camilli, P. (1994) Mol. Biol. Cell 5, 194 (abstr.)
  29. Carpenter, G. (1992) FASEB J. 6 , 3283-3289 [Abstract/Free Full Text]
  30. Vigers, G. P. A., Crowther, R. A., and Pearse, B. M. F. (1986) Eur. Mol. Biol. Org. J. 5, 2079-2085 [Abstract]
  31. Maycox, P. R., Link, E., Reetz, A., Morris, S. A., and Jahn, R. (1992) J. Cell Biol. 118, 1379-1388 [Abstract]
  32. Robinson, P. J., Sontag, J.-M., Liu, J.-P., Fykse, E. M., Slaughter, C., McMahon, H., and Südhof, T. C. (1993) Nature 365, 163-166 [CrossRef][Medline] [Order article via Infotrieve]

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