Serine 331 and Tyrosine 333 Are Both Involved in the Interaction between the Cytosolic Domain of TGN38 and the μ2 Subunit of the AP2 Clathrin Adaptor Complex*
- From the Department of Biochemistry, University of Bristol, University Walk, Bristol BS8 1TD, United Kingdom
Abstract
TGN38 is a type I integral membrane protein that cycles between the trans-Golgi network and the plasma membrane. Internalization at the cell surface and targeting back to thetrans-Golgi network is dependent on a hexapeptide motif, SDYQRL, in the cytosolic tail of the protein. It was recently demonstrated that this motif specifically interacts with the μ2 subunit of the AP2 adaptor complex. We have studied the interaction between the entire cytosolic domain of TGN38 and μ2 using the yeast two hybrid system, in vitro binding of recombinant fusion proteins and IAsys optical biosensor technology. A specific interaction has been demonstrated in each of the systems we have employed. We have shown an absolute requirement for Tyr-333 of TGN38 in binding to μ2. In addition we found that mutation of Ser-331 to alanine reduces the affinity of the interaction. By measuring tryptophan fluorescence at equilibrium, we have also determined the dissociation constant for the interaction between the entire cytosolic tail of TGN38 and μ2 as 58 nm. In contrast to previously published work, our data suggest that not only Tyr-333 but also its context is important in determining the specificity of binding of TGN38 to μ2.
Footnotes
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↵* This work was supported by the Medical Research Council and Biotechnology and Biological Sciences Research Council.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ These authors contributed equally to this work.
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↵§ To whom correspondence should be addressed. Tel.: 44-117-928-8272; Fax: 44-117-928-8274; E-mail: bantingg{at}bsa.bristol.ac.uk.
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↵1 The abbreviations used are: TGN,trans-Golgi network; Trx, thioredoxin A; GST, glutathioneS-transferase; PCR, polymerase chain reaction; X-gal, 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside.
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↵2 The Internet address ishttp://rsb.info.nih.gov/nih-image.
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↵3 E. P. Roquemore and G. Banting, submitted for publication.
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- Received November 27, 1996.
- Revision received March 31, 1997.
