Volume 8 issue 10 October 2007
Mucolipin-2 Localizes to the Arf6-Associated Pathway and Regulates Recycling of GPI-APs
Claudia Karacsonyi, Anitza San Miguel, and Rosa Puertollano*

*Corresponding author:

Rosa Puertollano. Laboratory of Cell Biology. National Heart, Lung, and Blood Institute. National Institutes of Health. Bethesda, Maryland 20892, USA. Tel.: +1 (301) 451-2361; FAX: +1 (301) 480-0357; E-mail: puertolr@mail.nih.gov

Running title: MCOLN2 Regulates Traffic of GPI-APs

Keywords: mucolipin, TRP, Arf6, GPI, MLIV, MCOLN2

Supplemental Figure 1: MCOLN2-RFP, CD59 and MHCI accumulate in ARF6Q67L-associated vacuolar endosomes. HeLa cells were transfected with plasmids encoding MCOLN2-RFP and ARFQ67L-HA. 15 h after transfection the cells were fixed, permeabilized and immunostained with antibodies to CD59 (A), MHCI and HA (B) or to CD59 and lamp-1 (C). Arrows indicate ARF6Q67L-positive vacuoles. Arrowheads point to lysosomes. Inset in (A) shows a two-fold magnification of the indicated region. Scale bar represents 10 µm.

Figure 1 (.jpg)

Supplemental Figure 2: MCOLN2 accumulates at Rab5Q79L-positive endosomes. Hela cells were transiently co-transfected with plasmids encoding MCOLN2-RFP and Rab5Q79L. 15 h after transfection cells were fixed and examined by confocal microscopy (A), or incubated with monoclonal antibodies against CD59 (B) or CD63 (C). Arrows indicate accumulation of MCOLN2-RFP in RabQ69L-positive vacuoles. Insets show a two-fold magnification of the indicated regions. Scale bar represents 10 µm.

Figure 2 (.jpg)

Supplemental Figure 3: Specificity of MCOLN2-induced activation of Arf6. Hela cells were transfected with: (A) Arf6wt-HA alone or together with MCOLN1-RFP or MCOLN2-RFP; (B) Arf6Q67L-HA alone or together with MCOLN2-RFP; (C) Arf1-HA alone or together with MCOLN2-RFP for 24h. Pull-down assays were carried out using GST or GGA1GAT-GST to isolate GTP-bound Arf6. Quantifications were done from immunoblots using the Odyssey Infrared System.

Figure 3 (.jpg)

Supplemental Figure 4: Effect of MCOLN2 depletion on CD59 trafficking. (A) Hela cells expressing control siRNA or specific siRNA against MCOLN2 were transfected with Lamp1-GFP, MCOLN1-GFP, MCOLN2-GFP or MCOLN3-GFP, lysated and resolved by 4-20% SDS-PAGE. Immunoblot analysis with antibodies against GFP revealed a band of approximately 90 KDa that correspond with predicted molecular weight for the monomeric forms of full length MCOLN1-GFP, MCOLN2-GFP, and MCOLN3-GFP fusion proteins (arrow). Asterisks correspond to putative proteolytic forms, while higher bands correspond to dimers and oligomers (arrow). (B) HeLa cells were transfected with control or MCOLN2 siRNAs. After 72h, cells were incubated at 37°C with monoclonal antibodies to CD59 for 30 min, washed with acidic buffer, placed at 37°C for 15 min to allow reappearance of internalized CD59 at the cell surface, fixed, incubated with secondary antibody, and analyzed by FACS. The graphic represents the average of three independent experiments with triplicates for each condition.

Figure 4 (.jpg)