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

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. Lipoprotein lipase (LPL)6 and hepatic lipase (HL) are secreted glycoproteins that hydrolyze triglycerides sequestered in the core of circulating lipoproteins. As such, they play important roles in lipoprotein remodeling and uptake. LPL also mediates tissue influx of fatty acids derived from triglyceride-rich lipoproteins, and both lipases have been implicated in atherosclerosis and inflammation (1-4). These functions require nascent lipase polypeptides to fold and assemble into native structures, a necessary prerequisite for enzyme activity and secretion. However, both lipases require a trans-acting factor to attain a functional state, as exemplified by a naturally occurring mutation in the mouse called combined lipase deficiency (cld). In tissues and cells homozygous for the cld mutation, intracellular levels of LPL mRNA and protein are normal; however, the vast majority of LPL protein (ϳ95%) remains within the endoplasmic reticulum (ER) as misfolded (inactive) mass that is eventually degraded (5-8). HL activity is also diminished in cld/cld cells but to a lesser extent than LPL (9). In affected newborn mice, the resulting combined lipase deficiency causes massive chylomicronemia and neonatal death because of tissue ischemia and the absence of triglyceride-derived fatty acid influx (10). Indeed, this phenotype mirrors an LPL knock-out model (11), indicating that cld causes virtual abolishment of LPL function. The identity of this critical lipase maturation factor was made clear when a gene encoding a predicted transmembrane protein on mouse chromosome 17 (Tmem112) was found to harbor the cld mutation (9). The gene was renamed lipase maturation factor 1 (Lmf1...

Lipase maturation factor 1 is required for endothelial lipase activity

“…Lipase proteins were then affi nity purifi ed from cell extracts, with Lmf1 copurifi cation assessed by immunodetection. As demonstrated previously ( 25 ), Lmf1 could be detected in association with LPL and HL but not with PL ( Fig. 3 ).…”

“…Thus, Lmf1 is a critical factor in the posttranslational maturation of nascent lipase polypeptides into active enzymes ( 24 ). Lmf1 is an ER membrane protein that has been shown to interact with LPL and HL through one of its loops extending into the ER lumen ( 25 ). Loss -of-function mutations (Y439X and W464X) have also been identifi ed in the human ortholog of Lmf1, LMF1 ( 22,26 ).…”

“…Lipase affi nity purifi cation and immunoblotting were performed as described previously ( 25 ). Before Western blot analysis of EL in cell culture medium, samples were concentrated by heparin-Sepharose affi nity purifi cation in batch mode.…”

“…Mouse Lmf1 cDNA was subcloned into the pcDNA3.1 expression vector (Invitrogen) containing an N-terminal c-Myc epitope tag ( 25 ).…”

Lipase Maturation Factor 1 (Lmf1) Is Induced by Endoplasmic Reticulum Stress Through Activating Transcription Factor 6α (Atf6α) Signaling

“…Nonetheless, Lmf1 is ubiquitously expressed in cells and tissues independent of the presence of lipases (8). Moreover, several naturally occurring splice variants of Lmf1 lack the domain that is critical for lipase maturation (14). Based on these observations, it has been hypothesized that in addition to its established function in the posttranslational maturation of lipases, Lmf1 may play a wider role in ER homeostasis (15).…”