|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
|
Fig. 7. The dileucine motif is crucial for the lysosomal targeting of β-catenin-uncoupled E-cadherin constructs. (A) Schematic representations of construct EC81 and its derivatives: EC81
MP14 and EC81MP39, EC81 derivatives with additional internal deletions of residues 602-615 or 578-616, respectively; EC20: a construct lacking 131 amino acids at its C-terminal. (B) Immunofluorescence images reveal that that the 20 amino-acid residues within the JM region of the E-cadherin cytoplasmic domain contain the signal for lysosomal targeting of β-catenin-uncoupled E-cadherin constructs. Cells expressing the indicated constructs were labeled with anti-HA mAbs. (C) TC and TE treatment revealed that a significant percentage of EC20 and EC81MP14 remained inside the cells. Cells expressing the indicated constructs were incubated with 0.01% trypsin for 10 minutes at 37°C in the presence of 2 mM Ca2+ (TC) or 1 mM EGTA (TE). Proteins were detected with anti-HA mAbs. Protein bands of high molecular mass (marked by asterisks) correspond to intracellular, incompletely processed proteins that retain the precursor segment. (D) The sequence of the 20 amino-acid residues in the JM region of the E-cadherin cytoplasmic domain and of those substituted in the mutant constructs. (E) Immunofluorescence images showing localization of the constructs. Cells expressing the indicated constructs were labeled with anti-HA mAb. EC81LA was detected at the cell surface, whereas other constructs were detected in the intracellular compartments. (F) Quantification of the intracellular pool of EC81 and its derivatives. Relative amounts of the intracellular pools obtained after TE treatment were quantified using NIH Image and expressed as a percentage of the pool of total protein obtained after TC treatment. Before trypsinization, cells were incubated for 30 minutes at 37°C in medium with or without 0.45 M sucrose (+suc or –suc, respectively). Inhibition of endocytosis by hypertonic medium did not change the intracellular pool of EC81, EC81PA and EC81DA but reduced that of EC81KR, EC81EPA and EC81LA.
|
