The Adipophilin C Terminus Is a Self-folding Membrane-binding Domain That Is Important for Milk Lipid Secretion

Brandi M Chong,Tanya D Russell,Jerome Schaack,David J Orlicky,Philip Reigan,Mark Ladinsky,James L Mcmanaman

doi:10.1074/jbc.m110.217091

Abstract

Cytoplasmic lipid droplets (CLD) in mammary epithelial cells undergo secretion by a unique membrane envelopment process to produce milk lipids. Adipophilin (ADPH/Plin2), a member of the perilipin/PAT family of lipid droplet-associated proteins, is hypothesized to mediate CLD secretion through interactions with apical plasma membrane elements. We found that the secretion of CLD coated by truncated ADPH lacking the C-terminal region encoding a putative four-helix bundle structure was impaired relative to that of CLD coated by full-length ADPH. We used homology modeling and analyses of the solution and membrane binding properties of purified recombinant ADPH C terminus to understand how this region possibly mediates CLD secretion. Homology modeling supports the concept that the ADPH C terminus forms a four-helix bundle motif and suggests that this structure can form stable membrane bilayer interactions. Circular dichroism and protease mapping studies confirmed that the ADPH C terminus is an independently folding α-helical structure that is relatively resistant to urea denaturation. Liposome binding studies showed that the purified C terminus binds to phospholipid membranes through electrostatic dependent interactions, and cell culture studies documented that it localizes to the plasma membrane. Collectively, these data provide direct evidence that the ADPH C terminus forms a stable membrane binding helical structure that is important for CLD secretion. We speculate that interactions between the four-helix bundle of ADPH and membrane phospholipids may be an initial step in milk lipid secretion.

Highlights

Adenoviral Expression of GFP-ADPH[1–220] in Mammary Epithelial Cells—Initial structure-function studies indicating that cytoplasmic lipid droplets (CLD) binding and stabilization functions of ADPH are located within its N-terminal half (Fig. 1A) [15,16,17] have led to speculation that the C-terminal portion of ADPH may contribute to CLD secretion by mediating interactions with elements of the plasma membrane [11]
Immunofluorescence and tomographic analyses of immunogold-stained cells stably expressing GFP-ADPH(fl)-VSV or GFP-ADPH[1–220]-VSV (Fig. 1B) demonstrate that these constructs correctly localize to the CLD surface and verify that the C-terminal region is not required for the CLD binding function of ADPH [15, 17]
We tested the importance of the ADPH C-terminal region in CLD secretion by determining the relative abilities of CLD coated with GFP-ADPH(fl)-VSV or GFP-ADPH[1–220]

Summary

EXPERIMENTAL PROCEDURES

Antibodies—Rabbit polyclonal antibodies specific to the C-terminal 15 amino acids (anti-ADPHcterm) or the N-terminal 25 amino acids (anti-ADPHnterm) of mouse ADPH were generated as described previously [19]. CLD Quantitation—Average CLD diameters were determined by analysis of individual CLD coated with GFPADPH(fl)-VSV, GFP-ADPH[1–220]-VSV, or endogenous ADPH in immunostained sections containing 60 – 80 randomly chosen alveoli at ϫ600 magnification as described previously [8]. Purified GST-ADPH[172– 425] was collected, rebound to glutathione-Sepharose, and digested with 50 units of thrombin (Sigma-Aldrich) to remove the GST tag. The purity of eluted protein was determined by SDS-PAGE and silver staining and immunoblot analysis using anti-ADPHcterm antibodies [19]. ADPH-bound liposomes were collected by centrifugation at 16,000 ϫ g for 15 min, resuspended in 100 ␮l of buffer, and analyzed by anti-ADPHcterm immunoblotting following SDS-PAGE. The optimal interaction of ADPH[172–425] with the membrane corresponded to the orientation with the minimum solvation energy

RESULTS

Residues residues helix helix Net charge

DISCUSSION