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. 2011 Apr 8;286(14):12602-16.
doi: 10.1074/jbc.M110.170217. Epub 2011 Jan 18.

BACE1 retrograde trafficking is uniquely regulated by the cytoplasmic domain of sortilin

Gina M Finan  1 , Hirokazu Okada, Tae-Wan Kim
Affiliations

BACE1 retrograde trafficking is uniquely regulated by the cytoplasmic domain of sortilin

Gina M Finan et al. J Biol Chem. .

Abstract

BACE1 (β-site β-amyloid precursor protein (APP)-cleaving enzyme 1) mediates the first proteolytic cleavage of APP, leading to amyloid β-peptide (Aβ) production. It has been reported that BACE1 intracellular trafficking, in particular endosome-to-TGN sorting, is mediated by adaptor complexes, such as retromer and Golgi-localized γ-ear-containing ARF-binding proteins (GGAs). Here we investigated whether sortilin, a Vps10p domain-sorting receptor believed to participate in retromer-mediated transport of select membrane cargoes, contributes to the subcellular trafficking and activity of BACE1. Our initial studies revealed increased levels of sortilin in post-mortem brain tissue of AD patients and that overexpression of sortilin leads to increased BACE1-mediated cleavage of APP in cultured cells. In contrast, RNAi suppression of sortilin results in decreased BACE1-mediated cleavage of APP. We also found that sortilin interacts with BACE1 and that a sortilin construct lacking its cytoplasmic domain, which contains putative retromer sorting motifs, remains bound to BACE1. However, expression of this truncated sortilin redistributes BACE1 from the trans-Golgi network to the endosomes and substantially reduces the retrograde trafficking of BACE1. Site-directed mutagenesis and chimera experiments reveal that the cytoplasmic tail of sortilin, but not those from other VPS10p domain receptors (e.g. SorCs1b and SorLA), plays a unique role in BACE1 trafficking. Our studies suggest a new function for sortilin as a modulator of BACE1 retrograde trafficking and subsequent generation of Aβ.

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Figures

FIGURE 1.
FIGURE 1.
Normalized expression levels of sortilin in the temporal cortex of patient-derived post-mortem brain tissue. A, human brain tissue from 12 control and 12 AD cases were analyzed via quantitative infrared Western blotting. The immunoblot was probed with sortilin, β-actin, TUJ1, and NeuN. B, plots displaying the immunoreactivity of sortilin antibody as normalized to β-actin, TUJ1, and NeuN for control versus AD brains. The horizontal line represents the normalized mean value in control versus AD subjects (*, p < 0.05).
FIGURE 2.
FIGURE 2.
Sortilin overexpression increases sAPPβ and secreted Aβ. A, HEK293 cells stably expressing either V5-tagged empty vector (vector) or V5-tagged sortilin (sort-V5) were compared via Western blotting. A, top two panels, representative data showing lysate subjected to SDS-PAGE and immunoblotted with anti-V5 or anti-APP. Full-length APP levels are shown (m, mature APP; im, immature APP). A, bottom two panels, immunoprecipitation of sAPPβ and Aβ from conditioned media. B, infrared quantification of immunoprecipitated sAPPβ. C, chemiluminescent quantification of immunoprecipitated total Aβ. Medium quantifications are normalized to total protein. Each bar represents the average of two experiments assayed in triplicate (lanes 1–3 of A). **, p < 0.01. Error bars, S.D.
FIGURE 3.
FIGURE 3.
Reducing sortilin decreases BACE1-mediated cleavage of APP. A, HEK293 cells were transiently transfected with either non-silencing shRNA vector (n.s.) or shRNA against human sortilin (shRNA). A, top two panels, representative Western blot analysis of sortilin down-regulation. β-Actin serves as a loading control. A, bottom three panels, immunoprecipitation of sAPPβ and Aβ from conditioned media. Full-length APP levels remain unchanged (m, mature APP; im, immature APP). B, chemiluminescent quantification of immunoprecipitated sAPPβ. C, chemiluminescent quantification of immunoprecipitated total Aβ. Medium quantifications are normalized to total protein. Each bar represents the average of two experiments assayed in duplicate. *, p < 0.05; **, p < 0.01. Error bars, S.D.
FIGURE 4.
FIGURE 4.
Co-immunoprecipitation analysis of exogenously and endogenously expressed sortilin and BACE1. A, Western blot analysis of co-immunoprecipitation input. HeLa cells were transiently co-transfected with sortilin (sort) and/or HA-tagged BACE1 (BACE1-HA). Negative controls include a mock transfection (no), sortilin transfection alone, and BACE1-HA transfection alone. B, co-immunoprecipitation (IP) results. Cells were lysed and subjected to co-immunoprecipitation using anti-sortilin or anti-HA. Samples were resolved via Western blotting using anti-HA and anti-sortilin, respectively. *, IgG. C, co-immunoprecipitation of HEK293 lysate by anti-BACE1 antibody. Negative controls include no antibody (beads) and a control antibody (mouse-derived) against an unrelated protein. Samples were resolved via Western blotting using anti-sortilin. D, human post-mortem brain tissue was co-immunoprecipitated with either no antibody (beads) or anti-BACE1. Samples were resolved via Western blotting using anti-sortilin.
FIGURE 5.
FIGURE 5.
A and B, truncated sortilin and BACE1 maintain binding affinity. A, co-immunoprecipitation (IP) of tagged full-length and truncated sortilin. HeLa cells were transiently co-transfected with either V5-tagged full-length sortilin (sort-V5), a cytoplasmic truncation of sortilin tagged with V5 (sortΔC-V5), and/or Myc-tagged BACE1 (BACE1-myc). Western blot analysis of input is depicted in the top panel. Negative controls include a mock transfection (no), sort-V5 alone, and sortΔC-V5 alone. The cells were lysed and subjected to immunoprecipitation using anti-Myc antibody. Samples were resolved via Western blotting using anti-V5. B, co-immunoprecipitation of untagged full-length and truncated sortilin. HeLa cells were transiently co-transfected with full-length sortilin (sort), a cytoplasmic truncation of sortilin (sortΔC), and/or HA-tagged BACE1 (BACE1-HA). Western blot analysis of input is depicted in the top panel. Negative controls include a mock transfection (no), sort alone, and sortΔC. The cells were lysed and subjected to immunoprecipitation using anti-HA. Samples were resolved via Western blotting using anti-sortilin. *, nonspecific band. C, truncated sortilin redistributes BACE1 from the TGN to peripheral punctate structures. C, left panels, distribution of full-length sortilin. HeLa cells were transiently transfected with sort-GFP and BACE1-CFP. Following fixation and permeabilization, the cells were stained with anti-syntaxin 6. C, right panels, distribution of truncated sortilin. HeLa cells were transiently transfected with sortΔC-GFP and BACE1-CFP. Following fixation and permeabilization, the cells were stained with anti-syntaxin 6. Magnification was ×100. D, truncated sortilin redistributes BACE1 from the TGN to the early endosomes. D, left panels, distribution of full-length sortilin. HeLa cells were transiently transfected with sort-GFP and BACE1-CFP. Following fixation and permeabilization, the cells were stained with anti-EEA1. D, right panels, distribution of truncated sortilin. HeLa cells were transiently transfected with sortΔC-GFP and BACE1-CFP. Following fixation and permeabilization, the cells were stained with anti-EEA1. Magnification was ×100.
FIGURE 6.
FIGURE 6.
Deletion of the cytoplasmic tail of sortilin leads to delayed retrograde trafficking of BACE1. A, following transient transfection of BACE1 in HEK293 cells stably expressing vector, sort-V5, and sortΔC-V5, BACE1 at the cell surface was labeled by N-terminal BACE1 antibody on ice (green). The labeled BACE1 was allowed to internalize at 37 °C and subsequently chased for the indicated time periods. Following fixation and permeabilization, the cells were stained with anti-TGN46 antibody (red). Representative confocal images are shown. Magnification was ×100. B, quantitative analysis using ImageJ image analysis software was performed to measure the co-localization of BACE1 with the TGN, revealing the percentage of BACE1 successfully delivered to the TGN (n = 5).
FIGURE 7.
FIGURE 7.
A, comparison of the cytoplasmic sorting motifs in Vps10p domain-sorting receptors. The luminal domains all contain at least one Vps10p domain. The sorting motifs in the short cytoplasmic tails are highlighted and differ between receptors. In the case of SorCS1b, no known sorting motifs have been identified. Their functions are indicated below the amino acid sequences. B, distribution of BACE1 with sortilin mutants in the early endosomes. HeLa cells were transiently co-transfected with BACE1-CFP along with either GFP-tagged full-length sortilin (sort), truncated sortilin (sortΔC), or various sortilin cytoplasmic motif double mutations. The mutation constructs harbor double mutations in the dileucine motif (L51A/L52A), a double mutation in the YSVL motif (Y14A/L17A), both of these alterations together (LL & YSVL), and a double mutation in the FLV motif (F9A/L10A). The cells were further stained with anti-EEA1 antibody (red) and analyzed via confocal microscopy. Magnification was ×100. C, distribution of BACE1 with sortilin mutants in the TGN. HeLa cells were transiently co-transfected with BACE1-CFP and either GFP-tagged full-length sortilin (sort), truncated sortilin (sortΔC), or various sortilin cytoplasmic motif double mutations. The mutation constructs harbor double mutations in the dileucine motif (L51A/L52A), a double mutation in the YSVL motif (Y14A/L17A), both of these alterations together (LL & YSVL), and a double mutation in the FLV motif (F9A/L10A). The cells were further stained with anti-TGN46 antibody (red) and analyzed via confocal microscopy. Magnification was ×100. TM, transmembrane domain.
FIGURE 8.
FIGURE 8.
A, schematic illustration of sortilin cytoplasmic domain chimeras. Two chimeric receptors harboring the luminal and transmembrane domains of sortilin and the cytoplasmic domains of either SorLA or SorCS1b. The constructs express GFP. B, analyses of BACE1 redistribution by sortilin chimeras in the early endosomes. Immunocytochemical analyses of sort-CS1b chimera and sort-SorLA chimera as compared with sortFL and sortΔC constructs. HeLa cells were transiently co-transfected with BACE1-CFP and either GFP-tagged sort, sortΔC, sort-CS1b chimera, or sort-SorLA chimera. Following fixation and permeabilization, cells were stained with anti-EEA1 and analyzed via confocal microscopy. Magnification was ×100. C, analyses of BACE1 redistribution by sortilin chimeras in the TGN. Immunocytochemical analyses of sort-CS1b chimera and sort-SorLA chimera as compared with sort and sortΔC constructs. HeLa cells were transiently co-transfected with BACE1-CFP and either GFP-tagged sort, sortΔC, sort-CS1b chimera, or sort-SorLA chimera. Following fixation and permeabilization, cells were stained with anti-TGN46 and analyzed via confocal microscopy. Magnification was ×100.
FIGURE 9.
FIGURE 9.
A, truncated sortilin decreases sAPPβ but leads to the accumulation of APP CTFs and increased Aβ. HEK293 cells stably expressing either V5-tagged empty vector (vector), V5-tagged sortilin (sort-V5), or V5-tagged truncated sortilin (sortΔC-V5) were compared. A, top three panels, representative data showing lysate subjected to SDS-PAGE and immunoblotted with anti-V5 or anti-APP. Full-length APP, C99 levels, and C89/C83 levels are shown. A, bottom two panels, immunoprecipitation of sAPPβ and Aβ from conditioned media. B, infrared quantification of immunoprecipitated sAPPβ. C, infrared quantification of immunoprecipitated C99. D, chemiluminescent quantification of immunoprecipitated total Aβ. Medium quantifications are normalized to total protein. Each bar represents the average of two experiments assayed in triplicate (lanes 1–3 in A). **, p < 0.01. E, truncated sortilin decreases sAPPβ and has the independent function of increasing C99 and Aβ by inhibiting CTF degradation. HEK293 cells stably expressing either V5-tagged empty vector (vector), V5-tagged sortilin (sort), or V5-tagged truncated sortilin (sortΔC) were compared for protein degradation. The left panels show untreated cells. The right panel shows cells treated with 75 μm chloroquine. Full-length APP was resolved via chemiluminescent Western blotting. Immunoprecipitated intracellular (arrow) and extracellular APPβ are shown in the second and third rows. Dark and light exposures of CTFs (arrowhead denotes C99) were resolved via quantitative infrared Western blotting.
FIGURE 10.
FIGURE 10.
Model for sortilin-mediated BACE1 trafficking. Trafficking of BACE1 by full-length sortilin. BACE1 and sortilin are endocytosed from the plasma membrane (PM) into the early endosome (EE). BACE1 gets retrogradely trafficked from the early endosome to the TGN by sortilin. We speculate that GGAs or retromer are involved in sortilin retrograde trafficking of BACE1. Sortilin cycles between the early endosome and the TGN. BACE1 is recycled to the plasma membrane via the TGN, forgoing degradation. Also depicted is the trafficking of BACE1 by truncated sortilin. BACE1 and sortilinΔC are endocytosed to the early endosome. Truncated sortilin is unable to retrogradely traffic BACE1 to the TGN, so sortilin and BACE1 trafficking are halted at the early endosome. It is likely that unphosphorylated BACE1 and truncated sortilin enter a direct recycling pathway, back to the plasma membrane, forgoing the TGN.
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