ZMBH J.Bericht 2000 - Zentrum für Molekulare Biologie der ...

Stefan Jentsch
Ubiquitin-Dependent Proteolysis
The central importance of selective proteolytic systems
in regulating cellular events has been recognized
recently. Progression through the eukaryotic cell
cycle, for example, is substantially regulated through
a timed and coordinated degradation of cyclins and
inhibitors of cyclin-dependent protein kinases. Similarly,
the shift from one transcriptional or developmental
program to another is often achieved through
a timed destruction of regulatory proteins. Proteolysis
is irreversible and therefore proteolytic enzymes are
usually employed for controlling unidirectional cellular
pathways. Selective degradation in eukaryotes is
primarily mediated by the ubiquitin/proteasome pathway.
Substrates of this pathway are covalently modified
by conjugation to ubiquitin, a small and highly
conserved protein. In most cases several ubiquitin
molecules are added to the substrate as a multiubi–
quitin chain in which ubiquitin molecules are arranged
like beads on a string. Multiubiquitinated proteins are
targeted for degradation by a large protease complex,
known as the 26S proteasome. Our research focuses
primarily on functional aspects of this pathway. We
identify from yeast and mammalian cells the enzymatic
components of this system, clone the corresponding
genes, and study their functions in vitro and
in vivo.
A novel ubiquitination factor, E4, involved in
multiubiquitin chain assembly
M. Kögl, T. Hoppe, S. Schlenker, H. D. Ulrich, T. U.
Mayer
Proteins modified by multiubiquitin chains are the
preferred substrates of the proteasome. Previous work
has suggested that E1, E2, and E3 enzymes are both
84
required and sufficient for the formation of multi–
ubiquitinated substrates. Recently, however, we could
show that efficient multiubiquitination needed for proteasomal
targeting of a model substrate (ubiquitin
fusions) requires an additional conjugation factor,
which we termed E4. This protein, previously known
as UFD2 in yeast, binds to the ubiquitin moieties of
preformed conjugates and catalyzes ubiquitin chain
assembly in conjunction with E1, E2, and E3. Intriguingly,
E4 defines a new protein family, which includes
two human members and the regulatory protein NOSA
from Dictyostelium required for fruiting body development.
In yeast E4 activity is linked to cell survival
under stress conditions, indicating that eukaryotes utilize
E4-dependent proteolysis pathways for multiple
cellular functions. Interestingly, UFD2 (E4) interacts
with CDC48, a member of the large family of AAAtype
ATPases. CDC48 is not involved in the ubiquitinconjugation
reaction but is needed for proteolysis of
some substrates of the ubiquitin/proteasome pathway.
Ubiquitin-like proteins from mammals and
yeast
D. Liakopoulos, G. Doenges
Ubiquitin is one of the most highly conserved eukaryotic
proteins known to date. Human ubiquitin differs
from its yeast homolog by only three amino acid residues
(out of 76 residues) and the proteins are functionally
equivalent. In addition to ubiquitin, two classes
of ubiquitin-related proteins have been identified. Proteins
of the first class carry ubiquitin-like domains
linked to unrelated sequences and are not conjugated
to other cellular proteins. One example is the mammalian
ubiquitin-related protein BAG-1 for which we
showed that it functions as a regulatory cofactor of
the Hsc70 chaperone. Proteins of the second class of
ubiquitin-like proteins are distinguished by their property
to become posttranslationally attached to other
cellular proteins analogously to ubiquitin. Known
members are SUMO-1 from higher eukaryotes and
its yeast ortholog SMT3. Recently, we have identified
a novel ubiquitin-like protein from yeast, which
we termed RUB1. RUB1 displays 53% amino acid
sequence identity to ubiquitin. We found that RUB1
conjugation to other cellular proteins requires at least
three proteins in vivo. ULA1 and UBA3 are related
to the amino- and carboxyl-terminal domains of the
E1 ubiquitin-activating enzyme, respectively, and together
fulfill E1-like functions for RUB1 activation.
RUB1 conjugation also requires UBC12, a protein
related to E2 ubiquitin-conjugating enzymes, which
functions analogously to E2 enzymes in RUB1-protein
conjugate formation. Conjugation of RUB1 is not
essential for normal cell growth and appears to be
selective for a small set of substrates. Remarkably, we
found that CDC53/cullin, a common subunit of the
multifunctional SCF ubiquitin ligase, is a major substrate
for RUB1 conjugation. This suggests that the
RUB1-conjugation pathway is functionally affiliated
with the ubiquitin/proteasome system and may play a
regulatory role.
Conjugation of the ubiquitin-like protein RUB1/
NEDD8 to cullin-2 is linked to von Hippel-Lindau
(VHL) tumor suppressor function
D. Liakopoulos, G. Doenges; in collaboration with T.
Büsgen, A. Brychzy, and A. Pause (Max Planck Institute
for Biochemistry, Martinsried)
Yeast RUB1 has homologs in higher eukaryotic cells.
The human RUB1 homolog, NEDD8, when expressed
in a yeast rub1 mutant, can complement its RUB1
deficiency. Recently we found that both hCUL-1
and hCUL-2 (homologs of yeast CDC53, see above)
are modified by the conserved ubiquitin-like protein
RUB1/NEDD8. Whereas hCUL-1 is part of a human
SCF complex, hCUL-2 assembles with elongin B/C
and the von Hippel-Lindau tumor suppressor protein
pVHL, forming a protein complex, CBC VHL , that
resembles SCF ubiquitin ligases. We could show that
NEDD8-hCUL-2 conjugates are part of CBC VHL complexes
in vivo. Remarkably, the formation of these
conjugates is stimulated by the pVHL tumor suppressor.
A tumorigenic pVHL variant, however, is
essentially deficient in this activity. Thus, ligation of
NEDD8 to hCUL-2 is linked to pVHL activity and
may be important for pVHL tumor suppressor function.
Moreover, our data indicate that modification of
cullins by NEDD8 requires the existence of a preassembled
ubiquitin ligase complex.
External Funding
During the period reported our research was supported
by grants from Deutsche Forschungsgemeinschaft
(project grants, SFB 352, Schwerpunktprogramm
Ubiquitin/Proteasomsystem, Leibniz Program,
Graduiertenkolleg), European TMR Ubiquitin
Research Network, American Cancer Society, and
Fonds der Chemischen Industrie.
PUBLICATIONS
Finley et al., (1998). Unified nomenclature for subunits
of the Saccharomyces cerevisiae proteasome regulatory
particle. Trends Biochem. Sci. 23, 244-245.
Hauser, H.-P., Bardroff, M., Pyrowolakis, G., and
85