Loss-of-Function in the Delta^RF Allele Is Due to a Failure of the Delta Protein to Reach the Cell Surface

Abstract

Endocytosis of the Delta ligand presumably drives "trans-endocytosis" of Notch resulting in proteolytic activation of the receptor. This model is founded in Delta mutants that reportedly fail to undergo endocytosis. One such allele, Dl^RF, gives a temperature-sensitive loss-of-function, purported to be an endocytosis mutant. Here we show that in lineages of Delta-expressing larval neurons the Dl^RF protein fails to localize to neurites consistent with a failure in trafficking to the plasma membrane. We find that the Dl^RF allele carries two mutations, G305E and C348Y, in the extracellular epidermal growth factor-like (EGF) repeats 3 and 4. Co-expression of GFP-tagged Dl^RF and RFP-tagged wild-type Delta (Dl^WT) in S2 cells demonstrates complete segregation of the proteins at 30ºC with Dl^RF failing to localize to the plasma membrane. As well, Dl^RF fails to undergo a characteristic proteolytic processing. Altogether the data indicate that the Dl^RF allele has limited utility for studying Delta endocytosis mechanisms but is well suited as a tool for loss-of-function studies.