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 tetraspa-nin-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 struc-tures, are important for cell-surface tetraspanin expression. Moreover, we unravel that SPPL3, a Golgi intra-membrane-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 pre-sentation of tetraspanins with implications for strategies to manipulate their functions, including cancer cell invasion.