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

Area of Science:

• Molecular biology
• Cell biology
• Virology

Background:

• The influenza virus hemagglutinin (HA) protein plays a crucial role in viral entry and is subject to cellular trafficking pathways.
• Cell surface protein localization is often regulated by specific amino acid sequences within their cytoplasmic domains, influencing sorting and internalization.
• Tyrosine-based motifs are known regulators of protein trafficking, involved in both endocytosis and polarized sorting.

Purpose of the Study:

• To investigate whether tyrosine-based internalization signals in the influenza virus hemagglutinin (HA) cytoplasmic domain also function as basolateral sorting signals.
• To determine if the internalization and basolateral sorting functions associated with specific HA sequences can be genetically separated.
• To elucidate the distinct molecular requirements for internalization versus basolateral sorting mediated by HA cytoplasmic domain motifs.

Main Methods:

• Construction and analysis of influenza virus hemagglutinin (HA) mutants with specific amino acid substitutions (C543Y) and additions (HA+8).
• Utilizing Madin-Darby canine kidney (MDCK) cells, a standard model for studying epithelial cell polarity and protein trafficking.
• Introduction of second-site mutations into HA mutants to dissect the functional relationship between internalization and basolateral sorting signals.

Main Results:

• A mutation converting cysteine 543 to tyrosine (C543Y) in HA introduced both basolateral sorting and internalization signals.
• The HA+8 mutant, with eight additional amino acids including a potent internalization signal, was efficiently sorted to the basolateral surface.
• Mutational analysis demonstrated that while sequence positions could be important for both functions, internalization and basolateral sorting responded differently to amino acid changes, indicating distinct mechanisms for HA C543Y.
• For HA+8, the basolateral sorting signal relied on the same tyrosine as the internalization signal but exhibited different sequence sensitivities, further supporting distinct sorting processes.

Conclusions:

• Tyrosine-based internalization signals can function independently of basolateral sorting signals, despite potential sequence overlap.
• The influenza virus hemagglutinin cytoplasmic domain contains distinct signals that differentially regulate protein internalization and basolateral membrane targeting.
• Cellular sorting processes like internalization and basolateral targeting, even when utilizing overlapping or co-linear signals, are governed by distinct sequence characteristics and molecular mechanisms.