Tyrosine-dependent basolateral sorting signals are distinct from tyrosine-dependent internationalization signals

Converting cystine 543 to tyrosine in the influenza virus hemagglutinin (HA) introduces both a basolateral sorting signal and an internalization signal into the HA cytoplasmic domain. Another HA mutant, HA+8, contains eight additional amino acids at the end of the cytoplasmic domain that include a powerful internalization signal. HA+8 was also sorted efficiently to the basolateral surface of Madin-Darby canine kidney cells. The simplest explanation for the observation that multiple sorting phenotypes depend upon the same small amino acid sequence is that certain tyrosine-based internalization signals might also function as basolateral sorting signals. To test this hypothesis, second-site mutations were introduced into HA C543Y or HA+8 to determine if the internalization and basolateral sorting functions can be separated. For HA C543Y, the same sequence positions were important for both basolateral sorting and internalization, but the two functions responded differently to individual amino acid replacements, indicating that they were distinct. For HA+8, the basolateral sowing signal required the same tyrosine as the internalization signal, but did not share any other characteristics. Thus, even when basolateral sorting signals that depend on tyrosine overlap or are co-linear with internalizations signals, the two sorting processes are sensitive to different characteristics of the sequence.