Lipase Maturation Factor LMF1, Membrane Topology and Interaction with Lipase Proteins in the Endoplasmic Reticulum

Mark H Doolittle,Saskia B Neher,Miklós Péterfy,Osnat Ben-Zeev,Jo Ling-Liao,Ciara M Gallagher,Maryam Hosseini,Fen Yin,Howard Wong,Peter Walter,Miklos Peterfy

doi:10.1074/jbc.m109.049395

Abstract

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.

Highlights

Lipase maturation factor 1 (LMF1) is predicted to be a polytopic protein localized to the endoplasmic reticulum (ER) membrane
Its subcellular location seems ideally suited for such a role; lipase maturation factor 1 (Lmf1) is a type III
Three of the seven TM domains assigned by Membrane Organization Prediction Data (MemO) scored 100%, indicating that for both mouse and human LMF1 proteins, all eight methods were in complete agreement

Summary

Introduction

Lipase maturation factor 1 (LMF1) is predicted to be a polytopic protein localized to the endoplasmic reticulum (ER) membrane. All mouse and human LMF1 cDNA sequences, except for green fluorescent protein (GFP) fusions, were subcloned into the pcDNA3.1 expression vector (Invitrogen) containing an amino-terminal c-Myc epitope tag [9]. To limit possible models of LMF1 topology based on MemO prediction results, the cytoplasmic and/or ER lumenal orientation of the amino and carboxyl termini of LMF1 was determined.

Results

Conclusion