Multiple interactions drive adaptor-mediated recruitment of the ubiquitin ligase rsp5 to membrane proteins in vivo and in vitro.
Recognition of membrane proteins by the Nedd4/Rsp5 ubiquitin ligase family is a critical step in their targeting to the multivesicular body pathway. Some substrates contain "PY" motifs (PPxY), which bind to WW domains in the ligase. Others lack PY motifs and instead rely on adaptors that recruit the ligase to them. To investigate the mechanism of adaptor-mediated ubiquitination, we have characterized the interactions between the adaptor Bsd2, the ubiquitin ligase Rsp5, and the membrane proteins Cps1, Tre1, and Smf1 from Saccharomyces cerevisiae. We have reconstituted adaptor-mediated modification of Cps1 and Tre1 in vitro, and we show that two PY motifs in Bsd2 and two WW domains (WW2 and WW3) in Rsp5 are crucial for this. The binding of a weak noncanonical DMAPSY motif in Bsd2 to WW3 is an absolute requirement for Bsd2 adaptor function. We show that sorting of the manganese transporter Smf1, which requires both Bsd2 and Tre1, depends upon two PY motifs in Bsd2 and one motif in Tre1 but only two WW domains in Rsp5. We suggest that sequential assembly of first a Bsd2/Rsp5 complex, then a Tre1/Bsd2/Rsp5 complex followed by a rearrangement of PY-WW interactions is required for the ubiquitination of Smf1.
Sullivan
James A
JA
Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom.
Lewis
Michael J
MJ
Nikko
Elina
E
Pelham
Hugh R B
HR
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D056827:Endosomal Sorting Complexes Required for Transport; D008565:Membrane Proteins / Q000378:metabolism*; D008954:Models, Biological; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000166:cytology; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism; D043743:Ubiquitin-Protein Ligase Complexes / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D056827:Endosomal Sorting Complexes Required for Transport; D008565:Membrane Proteins; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D043743:Ubiquitin-Protein Ligase Complexes; C469287:RSP5 protein, S cerevisiae