Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Jan 7;111(1):267-72.
doi: 10.1073/pnas.1316482111. Epub 2013 Dec 16.

A mechanism for retromer endosomal coat complex assembly with cargo

Megan S Harrison  1 , Chia-Sui Hung, Ting-ting Liu, Romain Christiano, Tobias C Walther, Christopher G Burd
Affiliations

A mechanism for retromer endosomal coat complex assembly with cargo

Megan S Harrison et al. Proc Natl Acad Sci U S A. .

Abstract

Retromer is an evolutionarily conserved protein complex composed of the VPS26, VPS29, and VPS35 proteins that selects and packages cargo proteins into transport carriers that export cargo from the endosome. The mechanisms by which retromer is recruited to the endosome and captures cargo are unknown. We show that membrane recruitment of retromer is mediated by bivalent recognition of an effector of PI3K, SNX3, and the RAB7A GTPase, by the VPS35 retromer subunit. These bivalent interactions prime retromer to capture integral membrane cargo, which enhances membrane association of retromer and initiates cargo sorting. The role of RAB7A is severely impaired by a mutation, K157N, that causes Charcot-Marie-Tooth neuropathy 2B. The results elucidate minimal requirements for retromer assembly on the endosome membrane and reveal how PI3K and RAB signaling are coupled to initiate retromer-mediated cargo export.

Keywords: biochemical reconstitution; mass spectrometry; proteoliposome; sorting nexin.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Mutations on the β1 and β3 strands ablate function but not endosome localization of yeast Snx3. (A) Ribbon diagram of yeast Snx3. The location of mutations on β1 (magenta E37A E39A H41A) and β3 (green R74A K77A) are indicated, and the site of the Gly23Cys mutation used to derivatize Snx3 is indicated in red. The structure is based on coordinates published in Zhou et al (16). (B) In vivo localization of Snx3-GFP. Each mutant Snx3 gene replaced the wild-type locus and is expressed with a C-terminal GFP tag. (C) The Snx3- and retromer-dependent cargo, Ste13, was expressed as a GFP fusion in cells expressing the indicated form of Snx3 and imaged by deconvolution microscopy. For B and C, one image from the approximate center of a Z series is shown. (Scale bar: 5 μm.)
Fig. 2.
Fig. 2.
Mutations in β1 of yeast and human SNX3 perturb recognition by retromer cargo selective complex. (A) Coimmunoprecipitation of myc epitope-tagged Snx3 with Vps29-HA immunopurified from yeast cell lysates prepared in the presence of 5 mM DSP cross-linking reagent was visualized by anti-myc blot (Lower). Vps29-HA levels were visualized by anti-HA blot (Upper). (B) Human retromer was immobilized and incubated with purified, recombinant SNX3 or SNX3-β1 mutant. Bound fractions were assayed by anti-SNX3 immunoblot (Upper) and staining with CBB (Lower).
Fig. 3.
Fig. 3.
SNX3 is recognized by VPS35 retromer subunit. (A) SNX3-Mts-Atf-biotin cross-linking strategy: Assembled SNX3*–retromer complex was exposed to UV light to activate the Atf (X) crosslinkage. Snx3* was subsequently released by reduction. An aliquot of the reaction was removed and probed by streptavidin-HRP blotting. The remainder was subjected to proteolysis, and derivatized peptides were identified by coupled liquid chromatography high resolution mass spectrometry (LC/MS). (B) CBB-stained gels of the indicated reactions (Left). Streptavidin-HRP blots of the same fractions (Right). Anti-SNX3 immunoblots of the blotted fractions are shown below the streptavidin-HRP blot. The asterisk indicates the position of free GST. (C) Summary of LC/MS analysis of Mts-Atf-biotin cross-linked products. Portions of the spectrum containing the major cross-linked peptides, which are each derived from VPS35, are shown. The corresponding mass data are shown in the table. The underlined Lys residues were modified with biotin. (D) Schematic diagram of VPS35 and location of SNX3–cross-linked peptides. VPS35 is proposed to be composed of 17 helical repeats (18). The approximate positions of the minimal regions of VPS35 that bind VPS26, VPS29, and RAB7 (red) and modified VPS35 peptides (yellow) are indicated.
Fig. 4.
Fig. 4.
SNX3 and RAB7 bind retromer simultaneously. GST-RAB7 fusion proteins were immobilized on GSH beads: GST-RAB7 (native RAB7), RAB7-Q67L (GTP-bound mutant), and RAB7-T22N (GDP-bound mutant). A CBB stained gel (Top) anti-VPS26 blot (Middle), and anti-SNX3 blot (Bottom) are shown.
Fig. 5.
Fig. 5.
Dual recognition of SNX3 and RAB7 recruits retromer to a membrane. (A) Liposomes were preincubated with purified SNX3, SNX3-β1, and/or RAB7-Q67L-His, and then purified retromer was added. Liposomes were collected by centrifugation, and the supernatant (S) and pellet (P) fractions (equal amounts) were loaded onto gels. A CBB-stained gel (Upper) and anti-VPS26 blot (Lower) are shown. The proportions of VPS26 in the pellet fractions, determined from this blot, are indicated below it. (B) Liposomes were incubated with RAB7-Q67L-His (“RAB7GTP”) or RAB7-T22N-His (“RAB7GDP”) with and without wild-type SNX3. Cosedimentation of retromer was monitored as described above. (C) Liposomes were preincubated with RAB7GTP or RAB7GTP K157N, with or without SNX3, and purified retromer was added. Cosedimentation of retromer was monitored as described above. (D) Retromer sedimentation was assayed with proteoliposomes alone or preincubated with SNX3 and RAB7. The proteoliposomes contained a retromer sorting motif (Tyr-Leu-Met; Center) or a peptide with each residue of this sequence, and surrounding aromatic residues, substituted by alanine.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Seaman MN, McCaffery JM, Emr SD. A membrane coat complex essential for endosome-to-Golgi retrograde transport in yeast. J Cell Biol. 1998;142(3):665–681. - PMC - PubMed
    1. Seaman MN. The retromer complex - endosomal protein recycling and beyond. J Cell Sci. 2012;125(Pt 20):4693–4702. - PMC - PubMed
    1. Cullen PJ, Korswagen HC. Sorting nexins provide diversity for retromer-dependent trafficking events. Nat Cell Biol. 2012;14(1):29–37. - PMC - PubMed
    1. Nakada-Tsukui K, Saito-Nakano Y, Ali V, Nozaki T. A retromerlike complex is a novel Rab7 effector that is involved in the transport of the virulence factor cysteine protease in the enteric protozoan parasite Entamoeba histolytica. Mol Biol Cell. 2005;16(11):5294–5303. - PMC - PubMed
    1. Rojas R, et al. Regulation of retromer recruitment to endosomes by sequential action of Rab5 and Rab7. J Cell Biol. 2008;183(3):513–526. - PMC - PubMed

Publication types