Mechanosensitive channels are integral membrane proteins that sense mechanical 
stimuli. Like most plasma membrane ion channel proteins they must pass through 
biosynthetic quality control in the endoplasmic reticulum that results in them 
reaching their destination at the plasma membrane. Here we show that N-linked 
glycosylation of two highly conserved asparagine residues in the 'cap' region of 
mechanosensitive Piezo1 channels are necessary for the mature protein to reach 
the plasma membrane. Both mutation of these asparagines (N2294Q/N2331Q) and 
treatment with an enzyme that hydrolyses N-linked oligosaccharides (PNGaseF) 
eliminates the fully glycosylated mature Piezo1 protein. The N-glycans in the 
cap are a pre-requisite for N-glycosylation in the 'propeller' regions, which 
are present in loops that are essential for mechanotransduction. Importantly, 
trafficking-defective Piezo1 variants linked to generalized lymphatic dysplasia 
and bicuspid aortic valve display reduced fully N-glycosylated Piezo1 protein. 
Thus the N-linked glycosylation status in vitro correlates with efficient 
membrane trafficking and will aid in determining the functional impact of Piezo1 
variants of unknown significance.