Site‐specific functionality and tryptophan mimicry of lipidation in tetraspanin CD9

Lipidation of transmembrane proteins regulates many cellular activities, including signal transduction, cell–cell communication, and membrane trafficking. However, how lipidation at different sites in a membrane protein affects structure and function remains elusive. Here, using native mass spectrometry we determined that wild‐type human tetraspanins CD9 and CD81 exhibit nonstochastic distributions of bound acyl chains. We revealed CD9 lipidation at its three most frequent lipidated sites suffices for EWI‐F binding, while cysteine‐to‐alanine CD9 mutations markedly reduced binding of EWI‐F. EWI‐F binding by CD9 was rescued by mutating all or, albeit to a lesser extent, only the three most frequently lipidated sites into tryptophans. These mutations did not affect the nanoscale distribution of CD9 in cell membranes, as shown by super‐resolution microscopy using a CD9‐specific nanobody. Thus, these data demonstrate site‐specific, possibly conformation‐dependent, functionality of lipidation in tetraspanin CD9 and identify tryptophan mimicry as a possible biochemical approach to study site‐specific transmembrane‐protein lipidation. Lipidation of transmembrane proteins regulates many cellular activities, but whether this is site‐specific remains elusive. We revealed that lipidation of tetraspanin CD9 at three sites suffices for EWI‐F binding. Mutations to alanine reduced binding, whereas binding was rescued by tryptophans. Thus, we demonstrate site‐specific functionality of lipidation in CD9 and identify tryptophan mimicry as an approach to study site‐specific lipidation.