Abstract
In eukaryotes, integration of sterols into the vacuolar/lysosomal
membrane is critically dependent on the Niemann–Pick type C
(NPC) system. The system consists of an integral membrane
protein, called NCR1 in yeast, and NPC2, a luminal soluble protein
that transfers sterols to the N-terminal domain (NTD) of NCR1
before membrane integration. Both proteins have been implicated
in sterol homeostasis of yeast and humans. Here, we investigate
sterol and lipid binding of the NCR1/NPC2 transport
system and determine crystal structures of the sterol binding
NTD. The NTD binds both ergosterol and cholesterol, with nearly
identical conformations of the binding pocket. Apart from sterols,
the NTD can also bind fluorescent analogs of phosphatidylinositol,
phosphatidylcholine, and phosphatidylserine, as well as
sphingosine and ceramide. We confirm the multi-lipid scope of
the NCR1/NPC2 system using photo-crosslinkable and clickable
lipid analogs, namely, pac-cholesterol, pac-sphingosine, and pacceramide.
Finally, we reconstitute the transfer of pac-sphingosine
from NPC2 to the NTD in vitro. Collectively, our results support
that the yeast NPC system can work as versatile machinery for
vacuolar homeostasis of structurally diverse lipids, besides
ergosterol.
membrane is critically dependent on the Niemann–Pick type C
(NPC) system. The system consists of an integral membrane
protein, called NCR1 in yeast, and NPC2, a luminal soluble protein
that transfers sterols to the N-terminal domain (NTD) of NCR1
before membrane integration. Both proteins have been implicated
in sterol homeostasis of yeast and humans. Here, we investigate
sterol and lipid binding of the NCR1/NPC2 transport
system and determine crystal structures of the sterol binding
NTD. The NTD binds both ergosterol and cholesterol, with nearly
identical conformations of the binding pocket. Apart from sterols,
the NTD can also bind fluorescent analogs of phosphatidylinositol,
phosphatidylcholine, and phosphatidylserine, as well as
sphingosine and ceramide. We confirm the multi-lipid scope of
the NCR1/NPC2 system using photo-crosslinkable and clickable
lipid analogs, namely, pac-cholesterol, pac-sphingosine, and pacceramide.
Finally, we reconstitute the transfer of pac-sphingosine
from NPC2 to the NTD in vitro. Collectively, our results support
that the yeast NPC system can work as versatile machinery for
vacuolar homeostasis of structurally diverse lipids, besides
ergosterol.
Originalsprog | Engelsk |
---|---|
Artikelnummer | e202402990 |
Tidsskrift | Life Science Alliance |
Vol/bind | 8 |
Nummer | 1 |
Antal sider | 15 |
ISSN | 2575-1077 |
DOI | |
Status | Udgivet - jan. 2025 |