Cytosolic Delivery of Functional Ubiquitin

The proteostasis network involves complex protein signaling cascades. The tagging of proteins with ubiquitin is central to the degradation of cellular proteins, but understanding its exact role in processing proteins is complicated by the complexity and extent of its utilization within cells. Here, we describe the application of a traceless protein delivery strategy to effect the uptake of exogenous ubiquitin into the cytosol of human cells. We find that coadministration of the endosomolytic peptides L17E and, especially, L17ER₄ provides not only cytosolic access to ubiquitin but also its functional incorporation into endogenous proteins. By enabling the study of semisynthetic ubiquitin variants in the human cytosol, this strategy could advance the field of ubiquitin biology.