A family of tetraspans organizes cargo for sorting into multivesicular bodies.

The abundance of cell-surface membrane proteins is regulated by internalization and delivery into intralumenal vesicles (ILVs) of multivesicular bodies (MVBs). Many cargoes are ubiquitinated, allowing access to an ESCRT-dependent pathway into MVBs. Yet how nonubiquitinated proteins, such as glycosylphosphatidylinositol-anchored proteins, enter MVBs is unclear, supporting the possibility of mechanistically distinct ILV biogenesis pathways. Here we show that a family of highly ubiquitinated tetraspan Cos proteins provides a Ub signal in trans, allowing sorting of nonubiquitinated MVB cargo into the canonical ESCRT- and Ub-dependent pathway. Cos proteins create discrete endosomal subdomains that concentrate Ub cargo prior to their envelopment into ILVs, and the activity of Cos proteins is required not only for efficient sorting of canonical Ub cargo but also for sorting nonubiquitinated cargo into MVBs. Expression of these proteins increases during nutrient stress through an NAD(+)/Sir2-dependent mechanism that in turn accelerates the downregulation of a broad range of cell-surface proteins.