Jansen EJ , van Bakel NH , Olde Loohuis NF , Hafmans TG , Arentsen T , Coenen AJ , Scheenen WJ , Martens GJ .

	FIGURE 1. C-terminal fusion of GFP to Ac45 interferes with C-terminal antibody recognition. A, transgenes used to generate transgenic Xenopus with expression of intact-Ac45 C- or N-terminally fused to GFP specifically in the intermediate pituitary melanotrope cells. B, direct GFP fluorescence was observed only in the IL and not in the AL of the pituitary. C, intact-Ac45/GFP fusion protein was recognized by the anti-GFP antibody but not by the anti-Ac45-C antibody. The GFP/intact-Ac45 fusion protein was recognized by both antibodies. SP, signal peptide; CS, cleavage site; TM, transmembrane domain; pA, SV40 polyadenylation site.
	FIGURE 2. Transgene expression of GFP-Ac45 mutant proteins specifically in the Xenopus melanotrope cells. A, overview of transgenes used to express Ac45 mutant proteins fused to GFP in the Xenopus melanotrope cells. B, sagital cryosections of transgenic Xenopus pituitaries. Transgenic Ac45 mutant/GFP expression was directly viewed under a fluorescence microscope (green). Sections were stained with an anti-POMC antibody (red) showing coexpression of GFP and POMC in the intermediate pituitary melanotrope cells. C, Western blot analysis of NIL lysates with an anti-GFP showing the expression levels of the respective transgene products. Ten percent of a total NIL lysate was analyzed. The lane with the Ac45ΔCS NIL lysate was taken from a separate Western blot. GFP*, stable GFP moiety, probably resulting from Ac45Nterm mutant fusion protein breakdown. D, endoproteolytic processing efficiencies of the Ac45wt and Ac45ΔC proteins are presented as the ratio of the amount of Ac45N-term relative to the amount of intact-Ac45 and the ratio of the amount of Ac45N-term relative to the amount of Ac45ΔC, respectively. Shown are the means � S.E. (n = 4). Significant difference is indicated by ** (p < 0.01).
	FIGURE 3. Subcellular localization of GFP-Ac45mutant proteins in transgenic Xenopus melanotrope cells. A, Xenopus melanotrope cells were isolated from the transgenic pituitaries, cultured and examined with live imaging for GFP fluorescence. Note that in the active melanotrope cells the ER is situated near the plasma membrane (53). B, melanotrope cells were fixed and stained with the marker antibodies anti-NaK-ATPase (plasma membrane), anti-calnexin (for the ER) and anti-POMC (for secretory vesicles).
	FIGURE 4. Ac45 mutant proteins differentially affect endogenous V-ATPase localization. A�D, immunogold labeling of ultra-thin pituitary cryosections with anti-GFP antibody confirmed the expression of the GFP/Ac45 mutant transgene products at the plasma membrane of the transgenic Xenopus melanotrope cells. No labeling was found in wild-type cells. E�H, immunogold labeling with an anti-V1A antibody showed that in the cleaved-Ac45 and Ac45 ΔC transgenic melanotrope cells the endogenous V-ATPase is recruited to the plasma membrane but not in wild-type cells nor in transgenic cells expressing cleaved-Ac45ΔC. White arrow, smooth plasma membrane; black arrow, microvillar plasma membrane; N, nucleus; ER, endoplasmic reticulum.
	FIGURE 5. Ac45Nterm is secreted via the regulated secretory pathway. Wild-type (wt) and Ac45Nterm-transgenic NILs were pulse labeled for 30 min and chased for 180 min in the presence of various concentrations of apomorphine, as indicated. A, five percent of the total labeled NIL lysates and 20% of the incubation media were directly resolved by SDS-PAGE. B, NIL lysates and incubation media were incubated with an anti-Ac45-N antibody, and the immune complexes were immunoprecipititated using protein A-Sepharose and resolved by SDS-PAGE. Radioactive signals were visualized by autoradiography.
	FIGURE 6. Dopaminergic inhibition of peptide release is affected in cleaved-Ac45- and Ac45 ΔC-transgenic Xenopus melanotrope cells. Wild-type (wt) and transgenic NILs were pulse-labeled for 30 min and chased for 180 min in the presence of 0.1 μm apomorphine. NILs were lysed and 5% of the total lysates and 20% of the incubation media was directly analyzed by SDS-PAGE. The lane with the cleaved-Ac45 ΔC NIL lysate was taken from a separate gel. Radioactive signals were visualized by autoradiography.
	FIGURE 7. Excess of cleaved-Ac45 does not affect the inhibition of the spontaneous Ca2+-oscillations in cleaved-Ac45-transgenic Xenopus melanotrope cells. A, Ca2+-oscillations in wild-type (wt) and cleaved-Ac45-transgenic (tg) cells loaded with fura-2 are inhibited in the presence of 0.1 μm apomorphine and reappear after removal of the drug. B, in the presence of 0.1 μm apomorphine low-frequency Ca2+-oscillations occur in ∼25% of the wt and tg cells.

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