Ecm29
The proteasome has traditionally been viewed as a discrete
complex containing stoichiometric subunits that survive stringent purification
methods. Although conventionally purified proteasomes are relatively
homogeneous, a variety of proteins interact detectably with the proteasome.
Among these are ubiquitin-like proteins such as Rad23, ubiquitinating
enzymes, deubiquitinating enzymes, adaptor proteins, and cell-cycle regulators.
Mass spectrometry and two-hybrid screens have been used to survey proteins
that interact with proteasomes. Taken together, these studies have suggested
that a diverse group of proteins interacts with the proteasome. However,
the functional significance of these interactions remains for the most
part poorly understood, and the results obtained using different methods
are not in close agreement. In some cases it is unclear whether interactions
identified through screens are also seen with intact proteasomes, and
in other cases it is unclear whether a significant fraction of proteasomes
are associated with a given factor. The group of Daniel Finley (Harvard
Medical School) has affinity-purified proteasomes from S.
cerevisiae and identified proteins associated with the proteasome purified in this
way.
Affinity-purified
proteasomes contain abundant proteins that are absent from proteasomes
purified in the conventional way, because elevated salt concentrations
dissociate them during purification. While many proteins may associate
with proteasomes transiently or in low amounts, only three - Ecm29,
Hul5, and Ubp6 - appeared to be major components. A clear role in regulating
proteasome activity was observed for Ecm29 and Ubp6. Ecm29 enhances
the
stability of the proteasome, whereas Ubp6 contributes to its enzymatic
activity. In addition, proteasomes dramatically activate Ubp6. These
results suggest that Ubp6 may help to release ubiquitin from proteasome-bound
conjugates, thus preventing translocation of ubiquitin onto the CP.
These
results suggested that the proteasome, as previously defined, represents
a core complex of salt-resistant subunits that is functionally distinct
from the complex responsible for protein degradation in
vivo.
) One of the major components of the affinity-purified proteasome
was the uncharacterized protein Ecm29, which had previously been
implicated in cell wall biogenesis. Proteasomes lacking Ecm29 were
prone to dissociation of the RP from the CP, indicating a role for
Ecm29 in stabilizing the CP-RP association. Electron micrographs
of free Ecm29 revealed a V-shaped morphology, in which the angle
between the two domains was not strictly fixed (top panels). Ecm29
complexed with the CP frequently displayed an open V-shaped morphology
as well (lower panels). In these images, Ecm29 projected from the
CP in the same direction as the RP, suggesting that CP-bound Ecm29
could contact the RP. Indeed, Ecm29 can bind both CP and RP, which
may be sufficient to explain its role in stabilizing the holoenzyme. |
 » Leggett et
al. (2002) Mol. Cell 10: 495-507. |