MLN64 Transport to the Late Endosome Is Regulated by Binding to 14-3-3 via a Non-canonical Binding Site

MLN64 is an integral membrane protein localized to the late endosome and plasma membrane that is thought to function as a mediator of cholesterol transport from endosomal membranes to the plasma membrane and/or mitochondria. The protein consists of two distinct domains: an N-terminal membrane-spanning domain that shares homology with the MENTHO protein and a C-terminal steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain that binds cholesterol. To further characterize the MLN64 protein, full-length and truncated proteins were overexpressed in cells and the effects on MLN64 trafficking and endosomal morphology were observed. To gain insight into MLN64 function, affinity chromatography and mass spectrometric techniques were used to identify potential MLN64 interacting partners. Of the 15 candidate proteins identified, 14-3-3 was chosen for further characterization. We show that MLN64 interacts with 14-3-3 in vitro as well as in vivo and that the strength of the interaction is dependent on the 14-3-3 isoform. Furthermore, blocking the interaction through the use of a 14-3-3 antagonist or MLN64 mutagenesis delays the trafficking of MLN64 to the late endosome and also results in the dispersal of endocytic vesicles to the cell periphery. Taken together, these studies have determined that MLN64 is a novel 14-3-3 binding protein and indicate that 14-3-3 plays a role in the endosomal trafficking of MLN64. Furthermore, these studies suggest that 14-3-3 may be the link by which MLN64 exerts its effects on the actin-mediated endosome dynamics

MLN64-interacting proteins identified by mass spectrometry.

<p>MLN64-interacting proteins identified by mass spectrometry.</p

MLN64 interacts with 14-3-3 <i>in vivo</i>.

<p>A) Western blot analysis of endogenous MLN64 from cell lysates immunoprecipitated with an antibody that recognizes all seven 14-3-3 isoforms indicates that MLN64 and 14-3-3 interact <i>in vivo</i>. The small amount of protein precipitated with the Rab5 antibody most likely represents a population in transit to the late endosome. B) Human cell extracts were immunoprecipitated using anti-14-3-3, anti-MLN64 or anti-vimentin antibodies. Precipitates were analyzed by Western blot probed for the presence of 14-3-3 proteins.</p

Expression of mutant MLN64 causes aberrant MLN64 and lipid trafficking in cells.

<p>A) Expression of either Wt or mutant MLN64 protein (red) in cells results in some co-localization with CD63-YFP (green); however, Wt MLN64 appears primarily vesicular, whereas mutant MLN64 exhibits more plasma membrane staining. Nuclei (blue) were stained with Hoechst 33342. B) Expression of either Wt or mutant MLN64 protein (green) results in accumulation of free cholesterol (blue) and GM1 (red) in endocytic vesicles. Cells transfected with mutant MLN64 accumulated more cholesterol than cells transfected with Wt MLN64; however, the levels of GM1 in both cell types were the equivalent. C) Quantitation of cholesterol accumulation in cells expressing either Wt MLN64 (Wt) or mutant MLN64* (Mut). AU; arbitrary units.</p

Lack of 14-3-3 binding causes delayed trafficking of MLN64 to the endosomal system.

<p>A) Huh7 cells transfected with MLN64-Flag (Wt) or MLN64*-Flag (Mut) for 24 hours were incubated at 20°C for 2 hr to cause accumulation of the protein at the Golgi. Immediately preceding release from the block, MLN64 proteins (green) exhibited a perinuclear staining pattern characteristic of the Golgi (t  =  0). Shortly after release from the block, Wt MLN64 is found in both vesicular compartments and the plasma membrane (t = 15, t = 30), in contrast to mutant MLN64, which remains primarily in the Golgi at 15 minutes. Only a small population of mutant protein appears on the plasma membrane and in vesicles at 30 minutes. Nuclei were counterstained with Hoechst 33342 (blue signal). B) MLN64-positive late endosomes (red) exhibit dissimilar localization in Huh7 cells transfected with DIFOPEIN-YFP or YFP alone (green) for 24 hrs. In YFP-expressing cells, MLN64 exhibits a characteristic perinuclear distribution, whereas in cells expressing DIFOPEIN-YFP, in which 14-3-3-ligand interactions are blocked, MLN64 recedes to the cell periphery. C) Co-expression of YFP (green) and MLN64-Flag (red) in Huh7 cells does not affect the normal perinuclear distribution of endosomes. In contrast, co-expression of DIFOPEIN-YFP with MLN64-Flag causes MLN64-positive vesicles to disperse toward the cell periphery, similarly to the effect seen in B. D) Huh7 cells transfected with MLN64-Flag and either DIFOPEIN-YFP or YFP alone were subjected to a temperature block at 20°C as described in A. At t = 0, MLN64 exhibits the perinuclear staining pattern that is characteristic of the Golgi. After release from the block, cells transfected with DIFOPEIN-YFP are localized mainly at the plasma membrane, with a small population being localized to vesicular compartments (t = 30). In contrast, cells transfected with YFP alone localize mainly in vesicular compartments, with a small population located at the plasma membrane.</p

Identification of MLN64-interacting proteins.

<p>A) Proteins bound to a START-affinity column were eluted with 0.1–1.5 M NaCl and resolved by gel electrophoresis. Proteins that remained associated at 0.4 M NaCl were isolated from the gel and identified by mass spectrometry. B) The 0.4 M NaCl elution fraction was also subjected to a blot overlay experiment using recombinant START protein to confirm potential binding partners.</p

Expression studies of full length and truncated MLN64 protein.

<p>A) MLN64 is a late endosomal protein that consists of four transmembrane segments and a cytosolic START domain. B) Schematic of full length and truncated MLN64 proteins containing only the transmembrane domains (TM) or the cytosolic START domain (START). C) Expression of MLN64, TM or START (red signal) causes accumulation of free cholesterol (blue signal) in the E/L system but only MLN64 and TM colocalize in late endosomes with CD63-YFP (green signal), whereas START displays an expression pattern characteristic of the ER. D) Cells expressing MLN64 and TM, but not START, display enlarged endosomal vesicles at 24 hours post-transfection, and this phenotype becomes more pronounced after 72 hours.</p

The MLN64 START domain is important for interactions with 14-3-3.

<p>A) An atypical 14-3-3 binding site at position 392 of the START domain of MLN64. B) According to a ribbon diagram of the crystal structure of the START domain, the predicted 14-3-3 binding site lies in an exposed loop (yellow) on the surface of the protein. C) Immunoprecipitation and western blot analysis of lysates from cells transfected with either MLN64-Flag (Wt) or MLN64*-Flag (Mut) indicates that the mutant MLN64 protein displays greatly reduced binding to the η isoform relative to the Wt protein. ε* denotes a 14-3-3 isoform that cannot bind target proteins. D) Similar to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034424#pone-0034424-g003" target="_blank">Figure 3A</a>, Western blot analysis of expressed mutant MLN64*-Flag from cell lysates bound to purified GST-tagged 14-3-3 isoforms indicates little or no interaction of mutant MLN64* and 14-3-3 γ, ε, η, ζ, τ and σ isoforms.</p

MLN64 interacts with 14-3-3 <i>in vitro</i>.

<p>A) Western blot analysis of endogenous MLN64 from cell lysates bound to purified GST-tagged 14-3-3 isoforms indicates interactions between MLN64 and 14-3-3 γ, ε, η, ζ, τ and σ isoforms. ε* denotes a 14-3-3 mutant that cannot bind target proteins. B) Western blot analysis of purified Flag-tagged MLN64 bound to purified 14-3-3-GST isoforms indicates the strongest interactions with the 14-3-3 η and τ isoforms.</p