This treatment resulted in the incorporation of Hrd1 into these vesicles in a uniform orientation with the catalytic RING (really interesting gene) domain facing the outside. When the vesicles were further incubated with ATP and ubiquitination factors, substrates of both topologies were polyubiquitinated, but only the luminally-oriented CPY*-TM population requires the Hrd1 trans membrane segments for ubiquitination. Because the reactions contain only Hrd1 and CPY -TMs, Hrdi apparently can move at least a portion of the luminally-localized CPY -TMs across the membrane using a conduit formed by its transmembrane domain.