of the Max - MDC

Intracellular Proteolysis
Thomas Sommer
Protein biogenesis is a remarkably imperfect process. About
one third of all newly synthesized proteins are presumably
defective. Functional proteins that were damaged by heat,
oxidizing conditions or toxic agents further increase the pool of
aberrant polypeptides. Defective proteins are toxic to cells and
must be properly taken care of. Accordingly, cellular chaperones
identify and repair deviant proteins. When salvage is not
possible, the ubiquitin-proteasome pathway (Figure 1) eliminates
the faulty elements. These so-called protein quality control
(PQC) pathways are found in most cellular compartments. A
major PQC pathway is found in the Endoplasmic Reticulum (ER)
and prevents the accumulation of malfolded or unassembled
proteins in the secretory pathway. Dysfunctions in this system
lead to severe diseases and, in addition, some viruses highjack
this system to establish themselves in the infected cell.
ER-associated protein degradation (ERAD) is an important
part of the PQC system of the ER. It can be divided mechanistically
into separate steps: First, misfolded proteins are
detected within the ER-lumen, a step that most likely
requires molecular chaperones as well as lectins that recognize
N-linked glycans. Second, proteolytic substrates are
targeted to and inserted into an aqueous transport channel,
which remains to be identified. Third, substrates are transported
back into the cytosol in a process termed protein dislocation.
Fourth, a polyubiquitin chain is synthesized on the
dislocated substrates. This step requires the action of membrane-bound
components of the ubiquitin system. In yeast
these are the ubiquitin-conjugating enzymes Ubc1, Ubc6
and Cue1 assembled Ubc7 and the ubiquitin ligases
Hrd1/Der3 and Doa10. Fifth, the AAA-ATPase Cdc48/p97
mobilizes ubiquitin-conjugated substrates. Finally, ub-binding
proteins help to transfer the substrates to the cytosolic
26S-proteasome for digestion.
Central to ERAD are ubiquitin ligases that are embedded in
the ER-membrane. Recently, we have characterized two central
ligase complexes on a molecular level and identified
novel components with unexpected new functions. In particular,
we have identified an ER-anchoring factor for the
AAA-ATPase (Cdc48/p97/VCP). This Cdc48 receptor in the
ER-membrane is called Ubx2 and associates with Hrd1/Der3
and Doa10. Moreover, we were able to show that the HRDligase
integrates protein quality control on the luminal site
of the ER-membrane with ubiquitin-conjugation and proteolysis
by the 26S proteasome on the cytosolic surface of this
compartment. Here, we have identified a novel factor, Yos9,
that seems to recognize specific oligosaccharide structures
on secretory proteins. Our current model of the function of
the HRD-ligase is depicted in Figure 2.
Ubx2 links the Cdc48/p97-Complex to
Endoplasmic Reticulum Associated Protein
Degradation
We and others have demonstrated that the AAA-ATPase
Cdc48/p97 plays a crucial role in protein dislocation.
However, the precise role in this transport step is not well
characterized. In addition, it remains to be clarified how
Cdc48/p97 is recruited to the ER-membrane. Using biochemical
approaches we were able to demonstrate that the
integral membrane protein Ubx2 mediates interaction of
the Cdc48/p97-complex with the ub-ligases Hrd1/Der3 and
Doa10. Ubx2 contains an UBX-domain that interacts with
Cdc48/p97 and an additional UBA-domain. Both domains
are located on the cytoplasmic surface of the ER and are
separated by two transmembrane segments. In cells lacking
Ubx2, the interaction of Cdc48/p97 with the ligase complexes
is abrogated and in turn breakdown of ER-proteins is
affected. Thus, protein complexes comprising the AAA-
ATPase, the recruitment factor Ubx2, and one of the known
ERAD ubiquitin ligases play central roles in ER-associated
proteolysis. Furthermore, degradation of a
cytosolic/nuclear protein, which is ubiquitinated by Doa10,
is disturbed in absence of Ubx2. This demonstrates that different
Cdc48/p97 dependent pathways converge at the ERsurface.
The Hrd1 ligase complex – a linchpin between
ER-luminal substrate selection and cytosolic
Cdc48 recruitment
A central ERAD component is the ubiquitin ligase
Hrd1/Der3. We have recently developed methods to study
extensively the interactions of yeast ER-membrane proteins
by co-immunoprecipitation and co-purification. Using this
assay, we were able to describe a complex of Hrd1/Der1 and
its partner protein Hrd3 with the ER-membrane protein
Der1. Our data imply that Hrd3 is the major substrate receptor
of this heterogenic ligase complex in the ER-lumen.
98 Cancer Research