1. Volume 10, Issue 3, Pages 343-354 (March 2006)
A Lumenal Domain-Dependent Pathway for Sorting to Intralumenal Vesicles of Multivesicular Endosomes Involved in Organelle Morphogenesis 
Alexander C. Theos, Steven T. Truschel, Daniele Tenza, Ilse Hurbain, Dawn C. Harper, Joanne F. Berson, Penelope C. Thomas, Graça Raposo, Michael S. Marks 
Developmental Cell 
Volume 10, Issue 3, Pages (March 2006)
DOI: /j.devcel
Copyright © 2006 Elsevier Inc. Terms and Conditions

2. Figure 1
Overexpression of Hrs, Tsg101, or Vps4(K173A) Alters the Distribution of MART-1, but Not Pmel
(A–L) Representative images from IFM analysis of 1011-mel cells transfected 24 hr prior to acetone fixation with (A–C) myc-Rab27a as a control, (D–F) myc-Hrs, (G–I) HA-Tsg101, or (J–L) V5-hVps4b(K173A) and labeled for the (A and D) myc, (G) HA, or (J) V5 epitope tag, (B, E, H, and K) MART-1, or (C, F, I, and L) Pmel (with antibody NKI-beteb) and fluorochrome-conjugated secondary antibodies. Insets: 5× magnification of indicated regions as (D, G, and J) blue/green, (E, H, and K) green/red, and (F, I, and L) blue/red merged images. Note that acetone fixation, required to preserve MART-1 immunogenicity, reduces cytoplasmic labeling for V5-hVps4b(K173A) relative to formaldehyde fixation (Figure 2; Figures S2 and 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). The scale bar represents 28 μm.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

3. Figure 2
Overexpression of Hrs, Tsg101, or Vps4(K173A) Alters the Distribution of X-Ubq, but Not Pmel
(A–L) The experiment is the same as the one described in Figure 1, except that cells were fixed with formaldehyde and labeled for the (A, D, G, and J) epitope tag, (B, E, H, and K) X-Ubq, or (C, F, I, and L) Pmel. Note the accumulation of X-Ubq in (E) HPM (arrows) and (H and K) ring-like structures (inset, arrow) and the (F, I, and L) exclusion of Pmel from them. Insets: 5× magnification of indicated regions and merged channels, as in Figure 1. The scale bar represents 20 μm.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

4. 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 the MART-1 label is similar to that of untransfected cells (see Figure S5A).
(C and D) MNT-1 cells were transfected with Hrs or control siRNA and were analyzed by (C) IFM or (D) IEM with antibodies to Hrs and either MART-1 or Pmel as indicated. Transfected cells in (C) are indicated by an asterisk. Note the absence of label for MART-1 in Hrs-depleted cells ([C], top). (D) Note the labeling for Pmel (PAG15) over fibrils and ILVs in an Hrs-depleted cell.
The scale bars repesent 200 nm.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

5. 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 (Ba–Bc) Pmel K629R or (Bd–Bf) PTT and labeled for the (Ba and Bd) Pmel lumenal domain (with HMB-50), (Bb and Be) LAMP-1, or (Bc and Bf) both. Insets: 4× magnification of indicated regions. Note the labeling for both K629R and PTT (red) within LAMP-1-positive (green) structures. The scale bar represents 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). The scale bar represents 200 nm.
Developmental Cell  , DOI: ( /j.devcel )
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6. Figure 5 Distinct Lumenal Subdomains Are Required for Sorting to ILVs
(A) Schematic of the Pmel lumenal subdomains 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 (Ba–Bc) wt Pmel, (Bd–Bf) ΔNTR, (Bg–Bi) ΔPKD, (Bj–Bl) ΔRPT, and (Bm–Bo) ΔKLD labeled for (Ba, Bd, Bg, Bj, and Bm) Pmel, (Bb, Be, Bh, Bk, and Bn) LAMP-1, or (Bc, Bf, Bi, Bl, and Bo) both. Insets: 4× 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 with ΔNTR and ΔPKD. The scale bar represents 22 μm.
Developmental Cell  , DOI: ( /j.devcel )
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7. Figure 6 Ultrastructural Analysis of Pmel Deletion Mutants
(A–D) Ultrathin cryosections of HeLa cells expressing (A and B) ΔPKD, (C) ΔRPT, and (D) ΔKLD were immunogold labeled with (A, B, and D) HMB-45 or (C) HMB-50 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 (C) ΔRPT and (D) ΔKLD is predominantly observed on the internal membranes of MVBs, and it is less evident on the limiting membrane. The scale bars represent 200 nm.
Developmental Cell  , DOI: ( /j.devcel )
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8. 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 1 hr 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 coexpression of furin-HA. Note the faster-migrating Mβ′ of ΔKLD.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions