Lipase maturation factor LMF1, membrane topology and interaction with lipase proteins in the endoplasmic reticulum.
Lipase maturation factor 1 (LMF1) is predicted to be a polytopic protein localized to the endoplasmic reticulum (ER) membrane. It functions in the post-translational attainment of enzyme activity for both lipoprotein lipase and hepatic lipase. By using transmembrane prediction methods in mouse and human orthologs, models of LMF1 topology were constructed and tested experimentally. Employing a tagging strategy that used insertion of ectopic glycan attachment sites and terminal fusions of green fluorescent protein, we established a five-transmembrane model, thus dividing LMF1 into six domains. Three domains were found to face the cytoplasm (the amino-terminal domain and loops B and D), and the other half was oriented to the ER lumen (loops A and C and the carboxyl-terminal domain). This representative model shows the arrangement of an evolutionarily conserved domain within LMF1 (DUF1222) that is essential to lipase maturation. DUF1222 comprises four of the six domains, with the two largest ones facing the ER lumen. We showed for the first time, using several naturally occurring variants featuring DUF1222 truncations, that Lmf1 interacts physically with lipoprotein lipase and hepatic lipase and localizes the lipase interaction site to loop C within DUF1222. We discuss the implication of our results with regard to lipase maturation and DUF1222 domain structure.
Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, California 90095, USA. markdool@ucla.edu
Mark H
MH
Doolittle
Saskia B
SB
Neher
Osnat
O
Ben-Zeev
Jo
J
Ling-Liao
Ciara M
CM
Gallagher
Maryam
M
Hosseini
Fen
F
Yin
Howard
H
Wong
Peter
P
Walter
Miklós
M
Péterfy
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008049:Lipase / Q000235:genetics / Q000378:metabolism*; D008071:Lipoprotein Lipase / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018613:Microscopy, Confocal; D008954:Models, Biological; D009154:Mutation; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
C504173:LIPC protein, human; C546366:LMF1 protein, human; C546367:LMF1 protein, mouse; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D008049:Lipase; C495700:Lipc protein, mouse; D008071:Lipoprotein Lipase