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. 2006 Mar;10(3):343-54.
doi: 10.1016/j.devcel.2006.01.012.

A lumenal domain-dependent pathway for sorting to intralumenal vesicles of multivesicular endosomes involved in organelle morphogenesis

Alexander C Theos  1 , Steven T Truschel, Daniele Tenza, Ilse Hurbain, Dawn C Harper, Joanne F Berson, Penelope C Thomas, Graça Raposo, Michael S Marks
Affiliations

A lumenal domain-dependent pathway for sorting to intralumenal vesicles of multivesicular endosomes involved in organelle morphogenesis

Alexander C Theos et al. Dev Cell. 2006 Mar.

Abstract

Cargo partitioning into intralumenal vesicles (ILVs) of multivesicular endosomes underlies such cellular processes as receptor downregulation, viral budding, and biogenesis of lysosome-related organelles such as melanosomes. We show that the melanosomal protein Pmel17 is sorted into ILVs by a mechanism that is dependent upon lumenal determinants and conserved in non-pigment cells. Pmel17 targeting to ILVs does not require its native cytoplasmic domain or cytoplasmic residues targeted by ubiquitylation and, unlike sorting of ubiquitylated cargo, is insensitive to functional inhibition of Hrs and ESCRT complexes. Chimeric protein and deletion analyses indicate that two N-terminal lumenal subdomains are necessary and sufficient for ILV targeting. Pmel17 fibril formation, which occurs during melanosome maturation in melanocytes, requires a third lumenal subdomain and proteolytic processing that itself requires ILV localization. These results establish an Hrs- and perhaps ESCRT-independent pathway of ILV sorting by lumenal determinants and a requirement for ILV sorting in fibril formation.

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Figures

Figure 1
Figure 1. Overexpression of Hrs, Tsg101, or Vps4(K173A) alters the distribution of MART-1 but not Pmel
Representative images from IFM analysis of 1011-mel cells transfected 24 h prior to acetone fixation with myc-Rab27a as a control (a–c), myc-Hrs (d–f), HA-Tsg101 (g–i), or V5-hVps4b(K173A) (j–l) and labeled for the myc (a, d), HA (g) or V5 (j) epitope tag, MART-1 (b, e, h, k) or Pmel (c, f, i, l, using antibody NKI-beteb) and fluorochrome-conjugated secondary antibodies. Insets, 5X magnification of indicated regions as blue/green (d, g, j), green/red (e, h, k) and blue/red (f, i, l) merged images. Note that acetone fixation, required to preserve MART-1 immunogenicity, reduces cytoplasmic labeling for V5-hVps4b(K173A) relative to formaldehyde-fixation (Figs. 2, S2, S3). Note the accumulation of MART-1, but not Pmel, within large HPM in d and e (arrows) and the ring-like structures in g and h (arrowhead, insets). Bar 28 μm.
Figure 2
Figure 2. Overexpression of Hrs, Tsg101, or Vps4(K173A) alters the distribution of X-Ubq, but not Pmel
Experiment is the same as in Fig. 1, except that cells were fixed with formaldehyde and labeled for the epitope tag (a, d, g, j), X-Ubq (b, e, h, k) or Pmel (c, f, i, l). Note the accumulation of X-Ubq in HPM (e and arrows) and ring-like structures (h, k inset arrow) and the exclusion of Pmel from them (f, i, l). Insets, 5X magnification of indicated regions and merged channels as in Fig. 1. Bar, 20 μm.
Figure 3
Figure 3. Pmel, but not MART-1, is present on ILVs in Hrs-overexpressing or Hrs-depleted cells
A. Ultrathin cryosections of FACS-sorted, myc-Hrs-overexpressing 1011-mel cells were immunogold labeled for myc (PAG15) and Pmel (PAG10). Pmel is localized on internal membranes of Hrs-positive compartments (arrowheads, inset). B. The same cells were immunogold labeled for myc (PAG10) and MART-1 (PAG15). Note the label for MART-1 on the limiting membrane of Hrs-positive compartments. The density of MART-1 label is similar to that of untransfected cells (see Suppl. Fig. 5A). C. and D. MNT-1 cells were transfected with Hrs or control siRNA, and analyzed by IFM (C) or IEM (D) with antibodies to Hrs and either MART-1 or Pmel as indicated. Transfected cells in C are indicated by *. Note the absence of label for MART-1 in Hrs-depleted cells (C, top). D, note labeling for Pmel (PAG15) over fibrils and ILVs in a Hrs-depleted cell. Bars, 200 nm.
Figure 4
Figure 4. Lumenal determinants direct ILV localization of Pmel
A. Topological domain structure of WT Pmel, the K629R mutant, Tac, and PTT and TTP chimerae. Pmel (orange) and Tac (maroon) lumenal, transmembrane and cytoplasmic domains are indicated; yellow, the cytoplasmic K629R substitution. B. IFM analysis of HeLa cells expressing Pmel K629R (a–c) or PTT (d–f) and labeled for the Pmel lumenal domain (with HMB-50; a, d), LAMP-1 (b, e), or both (c, f). Insets, 4X magnification of indicated regions. Note labeling for both K629R and PTT (red) within LAMP-1-positive (green) structures. Bar, 22 μm. C. Ultrathin cryosections of HeLa cells expressing PTT were immunogold labeled for Pmel (HMB-45 – PAG10). Note the labeling on ILVs of MVBs (arrows). Bar, 200 nm.
Figure 5
Figure 5. Distinct lumenal subdomains are required for sorting to ILVs
A. Schematic of the Pmel lumenal sub-domains and corresponding single domain deletion constructs, ΔNTR, ΔPKD, ΔRPT and ΔKLD. Line, dibasic PC cleavage site (CS); numbers, residues at the borders of domains and deletions. B. IFM analysis of HeLa cells expressing WT Pmel (a–c), ΔNTR (d–f), ΔPKD (g–i), ΔRPT (j–l) and ΔKLD (m–o), labeled for Pmel (a, d, g, j, m), LAMP-1 (b, e, h, k, n) or both (c, f, i, l, o). Insets, 4X magnification of indicated regions. WT, ΔRPT and ΔKLD were detected with HMB-50 anti-Pmel together with mAb H4A3 anti-LAMP-1 and isotype-specific secondary antibodies; ΔNTR and ΔPKD lose reactivity with HMB-50 and were thus detected with HMB-45 relative to rabbit anti-LAMP-1. Note the LAMP-1-ringed donut structures with WT Pmel, ΔRPT and ΔKLD but not ΔNTR and ΔPKD. Bar, 22 μm.
Figure 6
Figure 6. Ultrastructural analysis of Pmel deletion mutants
Ultrathin cryosections of HeLa cells expressing ΔPKD (A,B), ΔRPT (C) and ΔKLD (D) were immunogold labeled with HMB-45 (A, B, D) or HMB-50 (C) and PAG-10. A and inset. ΔPKD labeling on tubular endosomal membranes. B. ΔPKD also labels the limiting membrane of vacuolar endosomes (arrowheads) and tubules emanating from them (arrow), as well as some internal membranes. Labeling for ΔRPT (C) and ΔKLD (D) decorates predominantly the internal membranes of MVBs and less on the limiting membrane. Bars, 200 nm.
Figure 7
Figure 7. Localization to ILVs is required for proprotein convertase processing
A. HeLa cells expressing WT or mutant Pmel were metabolically pulse-labeled and chased as indicated. Pmel was immunoprecipitated from cell lysates with C-terminally-directed αPep13h antibody, fractionated by SDS-PAGE (ΔKLD, 15% acrylamide; others, 12% acrylamide), and analyzed by PhosphorImager analysis. Upper panels: regions surrounding P1, P2 and Mα (arrows; see text). Lower panels: regions surrounding Mβ. Right, migration of molecular weight markers. Note the appearance of Mβ by 1h of chase for WT, ΔRPT and ΔKLD (Mβ’), but not for ΔNTR or ΔPKD (arrowheads). The deletion in ΔKLD is within Mβ, resulting in faster migration. Representative of four independent experiments. B. Immunoblot analysis of whole cell lysate of HeLa cells cotransfected with the indicated Pmel mutant and either empty vector (−) or furin-HA (+). Blots were probed with αPep13h to Pmel (top, middle) or reprobed with anti-HA (bottom) to detect furin-HA. Only the relevant portions of the gels are shown. Top, immature P1 bands of ΔPKD and ΔKLD (P1A) and faster-migrating P1 bands of ΔNTR and ΔRPT (P1B). Middle, Mβ levels relative to P1 in cells expressing ΔNTR and ΔPKD, but not ΔRPT or ΔKLD, are increased by co-expression of furin-HA. Note the faster migrating Mβ’ of ΔKLD.
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