Sequential genome-wide CRISPR-Cas9 screens identify genes regulating cell-surface expression of tetraspanins

Tetraspanins, a superfamily of membrane proteins, mediate diverse biological processes through tetraspanin-enriched microdomains in the plasma membrane. However, how their cell-surface presentation is controlled remains unclear. To identify the regulators of tetraspanin trafficking, we conduct sequential genome-wide loss-of-function CRISPR-Cas9 screens based on cell-surface expression of a tetraspanin member, TSPAN8. Several genes potentially involved in endoplasmic reticulum (ER) targeting, different biological processes in the Golgi apparatus, and protein trafficking are identified and functionally validated. Importantly, we find that biantennary N-glycans generated by MGAT1/2, but not more complex glycan structures, are important for cell-surface tetraspanin expression. Moreover, we unravel that SPPL3, a Golgi intramembrane-cleaving protease reported previously to act as a sheddase of multiple glycan-modifying enzymes, controls cell-surface tetraspanin expression through a mechanism associated with lacto-series glycolipid biosynthesis. Our study provides critical insights into the molecular regulation of cell-surface presentation of tetraspanins with implications for strategies to manipulate their functions, including cancer cell invasion. [Display omitted] •Identifying key regulators of tetraspanin trafficking by CRISPR-Cas9 screens•The role of N-glycosylation in cell-surface presentation of tetraspanins•Control of cell-surface tetraspanin expression by a SPPL3/B3GNT5 axis•MGAT1/2 and SPPL3 are potential targets for blocking cancer cell invasion Using sequential genome-wide loss-of-function CRISPR-Cas9 screens, Yang et al. identify key molecular regulators of cell-surface tetraspanin presentation. They unravel that N-glycosylation modification and biosynthesis of lacto-series glycolipids control the internalization of tetraspanins. Their findings constitute an avenue for modulating the diverse functions of tetraspanins, including cancer cell invasion.