Structural insight into the ESCRT-I/-II link and its role in MVB trafficking.
ESCRT (endosomal sorting complex required for transport) complexes orchestrate efficient sorting of ubiquitinated transmembrane receptors to lysosomes via multivesicular bodies (MVBs). Yeast ESCRT-I and ESCRT-II interact directly in vitro; however, this association is not detected in yeast cytosol. To gain understanding of the molecular mechanisms of this link, we have characterised the ESCRT-I/-II supercomplex and determined the crystal structure of its interface. The link is formed by the vacuolar protein sorting (Vps)28 C-terminus (ESCRT-I) binding with nanomolar affinity to the Vps36-NZF-N zinc-finger domain (ESCRT-II). A hydrophobic patch on the Vps28-CT four-helix bundle contacts the hydrophobic knuckles of Vps36-NZF-N. Mutation of the ESCRT-I/-II link results in a cargo-sorting defect in yeast. Interestingly, the two Vps36 NZF domains, NZF-N and NZF-C, despite having the same core fold, use distinct surfaces to bind ESCRT-I or ubiquitinated cargo. We also show that a new component of ESCRT-I, Mvb12 (YGR206W), engages ESCRT-I directly with nanomolar affinity to form a 1:1:1:1 heterotetramer. Mvb12 does not affect the affinity of ESCRT-I for ESCRT-II in vitro. Our data suggest a complex regulatory mechanism for the ESCRT-I/-II link in yeast.
Gill
David J
DJ
MRC Laboratory of Molecular Biology, Medical Research Council Centre, Cambridge, UK. djg38@mrc-lmb.cam.ac.uk
Teo
Hsiangling
H
Sun
Ji
J
Perisic
Olga
O
Veprintsev
Dmitry B
DB
Emr
Scott D
SD
Williams
Roger L
RL
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D056827:Endosomal Sorting Complexes Required for Transport; D011992:Endosomes / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000737:chemistry*; D028861:Nuclear Pore Complex Proteins / Q000737:chemistry / Q000378:metabolism; D029543:Nucleocytoplasmic Transport Proteins; D011485:Protein Binding; D021381:Protein Transport; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D033921:Vesicular Transport Proteins / Q000737:chemistry* / Q000378:metabolism; D014982:Xenopus laevis; D015003:Yeasts
D056827:Endosomal Sorting Complexes Required for Transport; D005656:Fungal Proteins; D046912:Multiprotein Complexes; C517100:Mvb12 protein, S cerevisiae; C104361:NPL4 protein, S cerevisiae; D028861:Nuclear Pore Complex Proteins; D029543:Nucleocytoplasmic Transport Proteins; D029701:Saccharomyces cerevisiae Proteins; C102954:VPS28 protein, S cerevisiae; D033921:Vesicular Transport Proteins; C491128:Vps36 protein, S cerevisiae