The Role of Phosphoinositide 3-Kinase in the Sorting and Transport of Newly Synthesized Tyrosinase-Related Protein-1 (TRP-1)  Hua Chen, Thomas G. Salopek, Kowichi Jimbow  Journal of Investigative Dermatology Symposium Proceedings  Volume 6, Issue 1, Pages 105-114 (November 2001) DOI: 10.1046/j.0022-202x.2001.00012.x Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 WTM induces newly synthesized TRP-1 enriched vesicles to form a tubular network and results in the appearance of swollen vesicles (macrovesicles) in a concentration-dependent manner. MeWo cells were treated with WTM at various concentrations for 1 h at 37°C: (a) untreated controls, (b) 0.01 µM, (c) 0.1 µM, (d) 1 µM, and (e) 5 µM. Slides were fixed with methanol/acetone and incubated with antibody to TRP-1 and followed by fluorescein-labeled goat antimouse IgG. Arrows indicate WTM-induced tubular structures, whereas arrowheads point to macrovesicles. Journal of Investigative Dermatology Symposium Proceedings 2001 6, 105-114DOI: (10.1046/j.0022-202x.2001.00012.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 WTM inhibits PI 3-kinase activity in vitro. MeWo cells lysates were immuno-precipitated with antibody to its p110 subunit. Proteins released from the washed protein A-sepharose beads were assayed for PI 3-kinase activity in the presence of various concentrations of WTM, [α-32P]ATP, and phosphatidylinositol as described in the Materials and methods. The lipid products were extracted and separated by TLC. Results are presented as percentage activity of that seen in untreated control samples. (a) PI3-kinase activity in the presence of WTM; (b) autoradiogram. Journal of Investigative Dermatology Symposium Proceedings 2001 6, 105-114DOI: (10.1046/j.0022-202x.2001.00012.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 WTM induces the formation of a novel TRP-1 positive compartment in a time-course fashion. MeWo cells were exposed to WTM (1 µM) for various times at 37°C: (a) untreated, (b) 15 min, (c) 30 min, and (d) 1 h. Arrows indicate the formation tubular network of TRP-1 enriched vesicles, and arrowheads point to swollen TRP-1 vesicles. Journal of Investigative Dermatology Symposium Proceedings 2001 6, 105-114DOI: (10.1046/j.0022-202x.2001.00012.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 The distribution of internalized immunoglobulins to TRP-1 is similar to the endogenous distribution TRP-1 in WTM-treated MeWo cells, suggesting that the WTM-induced vesicular compartment is part of the endosomal pathway. MeWo cells were incubated in the presence of 1 µM WTM for 30 min, followed by MoAb to TRP-1 (i.e., HMSA-5) for 5 min (a, b), 15 min (c, d), and 30 min (e, f). The distribution of internalized anti-TRP-1 antibodies was assessed using secondary rhodamine-labeled goat antimouse IgG (a, c, e). The distribution of endogenous TRP-1 was determined by incubating methanol/acetone fixed slides with primary antibody to TRP-1 (α-PEP1), followed by fluorescein-labeled goat antimouse IgG (b, d, f). Arrows indicate the tubular network of TRP-1-enriched vesicles, and arrowheads point to swollen TRP-1 vesicles. Journal of Investigative Dermatology Symposium Proceedings 2001 6, 105-114DOI: (10.1046/j.0022-202x.2001.00012.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 The WTM-induced swollen compartment appears to be related to the late endosomes/prelysosomes. MeWo cells were untreated (a, c, e, g, i, k) or treated with WTM (1 µM) (b, d, f, h, j, l) for 1 h at 37°C. Methanol/acetone-fixed cells were double-labeled with mouse MoAb to TRP-1 (a, b, e, f, i, j) (as detected with a rhodamine-labeled goat antimouse IgG), and with rabbit polyclonal antibodies to CI-M6PR (c, d), LAMP-1 (g, h), or CD63 (k, l) (as detected with fluorescein-labeled swine antirabbit IgG). Arrows indicate sample sites of colocalization of TRP-1 with the lysosomal markers, whereas arrowheads demonstrate representive sites of vesicular structures that do not colocalize. Journal of Investigative Dermatology Symposium Proceedings 2001 6, 105-114DOI: (10.1046/j.0022-202x.2001.00012.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 WTM does not affect the processing of TRP-1. In contrast, Brefeldin A (BFA) interferes with the glycosylation of TRP-1. MeWo cells were pulse-labeled with [35S] methionine for 5 min, and chased for various times either in the presence or absence of (a) WTM (1 µM) or (b) BFA (50 µg per ml). As WTM is unstable at 37°C, it was replenished every hour during the entire chase period. Cells were lyzed in RIPA buffer for 30 min at 4°C and centrifuged at 12 000 × g for 15 min. The postnuclear-supernatants were subjected to a preclearing process in a mixture of mouse serum agarose and protein-A sepharose for 2 h at 4°C. The precleared extracts were immuno-precipitated with anti-TRP-1 antibody and analyzed by SDS-PAGE and followed by fluorography. Journal of Investigative Dermatology Symposium Proceedings 2001 6, 105-114DOI: (10.1046/j.0022-202x.2001.00012.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Brefeldin A does not induce tubulo-vesicular structures or macrovesicles, and can either prevent or diminish the effects of WTM on MeWo cells. MeWo cells were (a) untreated, or exposed to (b) BFA alone (50 µg per ml) for 1 h, (c) BFA for 1 h followed by WTM (1 µM) for 1 h, or conversely (d) WTM for 1 h followed by BFA. Slides were fixed in methanol/acetone, stained with antibody to TRP-1 followed by fluorescein-labeled goat antimouse IgG, and processed as per above. Journal of Investigative Dermatology Symposium Proceedings 2001 6, 105-114DOI: (10.1046/j.0022-202x.2001.00012.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 The WTM-induced TRP-1 compartment is dependent on microtubules and GTPases. MeWo cells were (a) untreated, (b) pretreated with nocodazole (20 µg per ml) for 10 min, or (c) pretreated with AlF−4 (30 mM NaF + 50 µM AlCl3) for 10 min, followed by the addition of WTM for 30 min. Slides were processed for immuno-fluorescent microscopy as noted above. Journal of Investigative Dermatology Symposium Proceedings 2001 6, 105-114DOI: (10.1046/j.0022-202x.2001.00012.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 9 The WTM-induced TRP-1 compartment appears to require functioning vacuolar H+-ATPase. MeWo cells were incubated with (a) WTM (1 µM) for 30 min, followed by (b) Baf A1 (0.1 µM), or (c) chloroquine (50 µM), both for 30 min. Slides were processed for immuno-fluorescent microscopy as previously noted. Journal of Investigative Dermatology Symposium Proceedings 2001 6, 105-114DOI: (10.1046/j.0022-202x.2001.00012.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 10 TRP-1 processing in the presence of WTM requires an active proton pump and not simple luminal acidification. MeWo cells were pulse-labeled with [35S] methionine for 4 h at either 20°C or 37°C in the presence of (a) WTM (1 µM), followed by (b) bafilomycin A1 (0.1 µM), or (c) chloroquine (50 µM). Cells were lyzed, immuno-precipitated with anti-TRP-1 antibody, and analyzed by SDS-PAGE. Journal of Investigative Dermatology Symposium Proceedings 2001 6, 105-114DOI: (10.1046/j.0022-202x.2001.00012.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions