Sterols block binding of COPII proteins to SCAP, thereby controlling SCAP sorting in ER.
Sterols inhibit their own synthesis in mammalian cells by blocking the vesicular endoplasmic reticulum-to-Golgi transport of sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP), a sterol-sensing protein that escorts SREBPs. Unable to reach the Golgi, SREBPs are not processed by Golgi-resident proteases, and they fail to activate genes required for cholesterol synthesis. The current studies were designed to reveal whether sterols block SCAP movement by inhibiting synthesis of special vesicles dedicated to SCAP, or whether sterols block SCAP incorporation into common coat protein (COP)II-coated vesicles. Through immunoisolation, we show that SCAP-containing vesicles, formed in vitro, also contain vesicular stomatitis virus glycoprotein (VSVG) protein, a classic marker of COPII-coated vesicles. Sterols selectively block incorporation of SCAP into these vesicles without blocking incorporation of VSVG protein. We show that the mammalian vesicular budding reaction can be reconstituted by recombinant yeast COPII proteins that support incorporation of SCAP as well as VSVG into vesicles. Sterols block SCAP incorporation into vesicles by blocking Sar1-dependent binding of the COPII proteins Sec 23/24 to SCAP. These studies demonstrate feedback control of a biosynthetic pathway by the regulated binding of COPII proteins to an endoplasmic reticulum-to-Golgi transport protein.
Department of Molecular Genetics, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9046, USA. peter.espenshade@utsouthwestern.edu
Peter J
PJ
Espenshade
Wei-Ping
WP
Li
Daisuke
D
Yabe
D000818:Animals; D016466:CHO Cells; D002213:Capsid / Q000378:metabolism*; D006224:Cricetinae; D004705:Endocytosis; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000378:metabolism*; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013261:Sterols / Q000494:pharmacology*; D014759:Viral Envelope Proteins / Q000378:metabolism
C028391:G protein, vesicular stomatitis virus; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; C102196:SREBP cleavage-activating protein; D013261:Sterols; D014759:Viral Envelope Proteins; D049452:Green Fluorescent Proteins