TY - JOUR
T1 - The ubiquitin-associated (UBA) 1 domain of Schizosaccharomyces pombe Rhp23 is essential for the recognition of ubiquitin-proteasome system substrates both in vitro and in vivo
AU - Medina, Bethan
AU - Paraskevopoulos, Konstantinos
AU - Boehringer, Jonas
AU - Sznajder, Anna
AU - Robertson, Morag
AU - Endicott, Jane
AU - Gordon, Colin
PY - 2012/12/7
Y1 - 2012/12/7
N2 - The ubiquitin-proteasome system is essential for maintaining a functional cell. Not only does it remove incorrectly folded proteins, it also regulates protein levels to ensure their appropriate spatial and temporal distribution. Proteins marked for degradation by the addition of Lys(48)-linked ubiquitin (Ub) chains are recognized by shuttle factors and transported to the 26 S proteasome. One of these shuttle factors, Schizosaccharomyces pombe Rhp23, has an unusual domain architecture. It comprises an N-terminal ubiquitin-like domain that can recognize the proteasome followed by two ubiquitin-associated (UBA) domains, termed UBA1 and UBA2, which can bind Ub. This architecture is conserved up to humans, suggesting that both domains are important for Rhp23 function. Such an extent of conservation raises the question as to why, in contrast to all other shuttle proteins, does Rhp23 require two UBA domains? We performed in vitro Ub binding assays using domain swap chimeric proteins and mutated domains in isolation as well as in the context of the full-length protein to reveal that the Ub binding properties of the UBA domains are context-dependent. In vivo, the internal Rhp23 UBA1 domain provides sufficient Ub recognition for the protein to function without UBA2.
AB - The ubiquitin-proteasome system is essential for maintaining a functional cell. Not only does it remove incorrectly folded proteins, it also regulates protein levels to ensure their appropriate spatial and temporal distribution. Proteins marked for degradation by the addition of Lys(48)-linked ubiquitin (Ub) chains are recognized by shuttle factors and transported to the 26 S proteasome. One of these shuttle factors, Schizosaccharomyces pombe Rhp23, has an unusual domain architecture. It comprises an N-terminal ubiquitin-like domain that can recognize the proteasome followed by two ubiquitin-associated (UBA) domains, termed UBA1 and UBA2, which can bind Ub. This architecture is conserved up to humans, suggesting that both domains are important for Rhp23 function. Such an extent of conservation raises the question as to why, in contrast to all other shuttle proteins, does Rhp23 require two UBA domains? We performed in vitro Ub binding assays using domain swap chimeric proteins and mutated domains in isolation as well as in the context of the full-length protein to reveal that the Ub binding properties of the UBA domains are context-dependent. In vivo, the internal Rhp23 UBA1 domain provides sufficient Ub recognition for the protein to function without UBA2.
KW - Cell-Free System
KW - DNA-Binding Proteins
KW - Proteasome Endopeptidase Complex
KW - Protein Binding
KW - Protein Structure, Tertiary
KW - Schizosaccharomyces
KW - Schizosaccharomyces pombe Proteins
KW - Ubiquitin
U2 - 10.1074/jbc.M112.419838
DO - 10.1074/jbc.M112.419838
M3 - Article
C2 - 23038266
VL - 287
SP - 42344
EP - 42351
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 50
ER -