Abstract
Recognition of sorting signals within the cytoplasmic tail of membrane proteins by adaptor protein complexes is a crucial step in membrane protein sorting. The three known adaptor complexes, AP1, AP2, and AP3, have all been shown to recognize tyrosine- and leucine-based sorting signals, which are the most common sorting signals within membrane protein cytoplasmic tails. Although tyrosine-based signals are recognized by the micro-chains of adaptor complexes, the subunit recognizing leucine-based sorting signals is less clear. In this report we show by surface plasmon resonance that the two leucine-based sorting signals within the cytoplasmic tail of the invariant chain bind independently from each other to AP1 and AP2 but not to AP3. We also show that both motifs can be recognized by the micro-chains of AP1 and AP2. Moreover, by using monomeric as well as trimeric invariant chain constructs, we show that adaptor binding does not require trimerization of the invariant chain.
Highlights
In this report we show by surface plasmon resonance that the two leucine-based sorting signals within the cytoplasmic tail of the invariant chain bind independently from each other to AP1 and AP2 but not to AP3
A dynamin mutant led to accumulation of MHCII/invariant chain (Ii) at the plasma membrane [14], and each of the sorting motifs allowed a fast internalization from the PM [10, 15], suggesting an indirect transport route
The different Ii constructs were expressed in E. coli and purified using a Ni2ϩ-NTA-agarose matrix
Summary
Construction of Plasmids—Plasmid pET3a.Ii⌬TM-His [25] encodes a soluble C-terminal histidine-tagged Ii molecule lacking the transmembrane domain. After extensive washing with 10 column volumes of 50 mM Tris/HCl, pH 7.5, 20 mM imidazole, and 0.5 M NaCl 1 M NaCl 20 mM Hepes/KOH, pH 7.3, 90 mM KCl, and 2 mM MgCl2, the His-tagged Ii⌬TM molecules were eluted with 250 mM imidazole in the same buffer. The extract was adjusted to 20 mM Hepes/KOH, pH 7.3, 100 mM potassium acetate, 2 mM MgCl2 and concentrated to 1–2 mg/ml protein using ultrafiltration in the stirring cell with a 10-kDa cut-off filter (Amicon, Witten, Germany). This clathrin-coated vesicle extract was frozen in liquid nitrogen and stored at Ϫ80 °C. It should be noted that the above-described models allow the determination of rate constants without reaching equilibrium during the experimental cycle
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