Fig. 1. Mapping minimal regions of clathrin HC and LC interaction. (A) Yeast SFY526 cells were co-transformed with the indicated bovine HC fragments (in pGBT9) plus full-length bovine brain LCb or the LCb fragment 77–165 (in pACT2). Quantitative β-galactosidase (β-gal) assays were performed, and results are shown in units as the mean ± SD of triplicate determinations. Above the fragments tested, a diagram of full-length HC is delineated, indicating the terminal, distal, proximal, trimerization and extreme C-terminal domains (Txd + C). Black bars below the HC diagram indicate sites previously implicated in LC binding (1213–1313, 1438–1481 and 1513–1522) (; ). Hatched lines within each fragment highlight the boundaries of the minimal region in HC (1267–1522) required for LC binding, mapped by these studies. (B) SFY526 cells were co-transformed with bovine HC fragment 1204–1522 (in pGBT9) and the indicated bovine brain LCb fragments (in pGAD424). Results of liquid β-gal assays are shown in units as the mean ± SD of triplicate determinations. Above the fragments tested, a diagram of full-length bovine brain LCb is delineated, indicating the phosphorylation domain (P), conserved region (Cons), calcium-binding site (Ca), previously predicted HC-binding site (HC), neuron-specific insert (N) and calmodulin-binding site (Cam). Hatched lines within each fragment highlight the boundaries of the minimal region in LCb (90–157) required for HC binding, mapped by these studies. The β-gal units shown in (A) are generally higher than those in (B) because LC fragments were constructed in different prey vectors in which pACT2 (for A) has a higher expression level than pGAD424 (for B).