Ubiquitin conjugation is known to target protein substrates primarily
to degradation by the proteasome or via the endocytic route. Here we d
escribe a novel protein modification pathway in yeast which mediates t
he conjugation of RUB1, a ubiquitin-like protein displaying 53% amino
acid identity to ubiquitin. Mire show that RUB1 conjugation requires a
t least three proteins in vivo. ULA1 and UBA3 are related to the N-and
C-terminal domains of the E1 ubiquitin-activating enzyme, respectivel
y, and together fulfil E1-like functions for RUB1 activation. RUB1 con
jugation also requires UBC12, a protein related to E2 ubiquitin-conjug
ating enzymes, which functions analogously to E2 enzymes in RUB1-prote
in in conjugate formation, Conjugation of RUB1 is not essential for no
rmal cell growth and appears to be selective fur a small set of substr
ates. Remarkably CDC53/cullin, a common subunit of the multifunctional
SCF ubiquitin ligase, was found to be a major substrate for RUB1 conj
ugation. This suggests that the RUB1 conjugation pathway is functional
ly affiliated to the ubiquitin-proteasome system and mag play a regula
tory role.