Figure 2. Endocytic targeting of Bmper. (a) MECs pulsed with Bmper conditioned me- dia or puriﬁed Bmper were chased with cold media or media containing chloroquine for 2 h. (b) A Western analysis for internalized Bmper (N-terminal fragment) and activated Smad in MECs treated or pulsed with cleaved full- length rBmper (6 nM) and harvested over 2 h (top). After pulsing with Bmper, MECs were chased for 2 h with “cold” media lacking rBmper (middle) or with culture medium containing 200 μM chloroquine (bottom). (c) Graphi- cal representation of normalized intracellular Bmper levels from the cold chase compared with the chloroquine chase from panel b. (Fig. 1, a and b). At 37°C, Bmper internalization (as detected by its presence in cell lysates) steadily increased over time. How- ever, Bmper internalization was barely detected at 4°C, and the small amount of Bmper detected after 20 min of treatment at 4°C was nearly eliminated by MECs after 1 h of treatment. The disappearance of both the N and C terminus of Bmper over the time course of this experiment suggest that Bmper is not only internalized but may also be further processed or degraded in- tracellularly. To distinguish internalized Bmper and Bmp4 from their accumulation at the cell surface, we stripped the plasma membrane with an acid wash procedure after a pulse-chase ex- periment (Fig. S1 a, available at http://www.jcb.org/cgi/content/ full/jcb.200808064/DC1). This procedure had no effect on the detection of the internalized Bmp proteins in whole cell lysates and confirmed that the accumulation of Bmper in cell lysates is a result of endocytosis and not caused by increased cell surface affiliation. Bmper internalization was detected in endothelial cells, rat neonatal ventricular cardiomyocytes, and mouse em- bryonic fibroblasts (MEFs), but not by HEK-293 or COS7 cells (Fig. 1 b), indicating that the machinery required for Bmper in- ternalization is restricted to a limited range of cell types. We used fluorescence confocal microscopy to confirm the localization of Bmper in both untreated MECs and MECs treated with recombinant Bmper (Fig. 1, c–h). In untreated MECs, en- dogenous Bmper was detected at low levels with a distinct punc- tate perinuclear pattern. This more sensitive immunofluorescent staining likely represents accumulation of a low abundance of protein within the secretory pathway, as this form of Bmper was detectable at extremely low levels when using the same antibody for Western analysis. In contrast, a significant amount of Bmper accumulated near the plasma membrane after treating the MECs for 5 min with recombinant Bmper (Fig. 1 g). When MECs were chased with cold media (lacking recombinant protein), levels of intracellular Bmper returned to their endogenous levels (unpub- lished data), suggesting that internalized Bmper undergoes effi- cient degradation. When MECs treated with Bmper were chased with cold media containing the lysotropic agent chloroquine (Fig. 1 h), Bmper accumulated in large punctate vesicles that were clearly distinct in pattern and abundance from that seen with endogenous Bmper within the secretory pathway or after a 5-min pulse (Fig. 1 i). To explore the possibility that internalized Bmper under- goes lysosome-dependent degradation, as suggested by the ef- fects of chloroquine in our immunofluorescence experiments, we next compared the endocytic behavior of purified recombinant Bmper to Bmper secreted in conditioned media from HEK-293 cells transiently transfected with a plasmid encoding full-length Bmper. The HEK-293 conditioned media contained both full- length and “cleaved” Bmper, indicating that the secretory com- partment of these cells is capable of cleaving the propeptide of Bmper through intracellular pH-dependent catalysis (Thuveson and Fries, 2000; Ambrosio et al., 2008; Serpe et al., 2008) or that these cells might express a yet to be identified protease capable of cleaving Bmper. The recombinant Bmper used in these studies is nearly completely cleaved. MECs were pulsed with either of the two sources of Bmper for 2 h, and then chased without recombi- nant Bmper but with cold media or cold media containing chloro- quine for an additional 2 h (Fig. 2 a). Both recombinant Bmper (6 nM) and Bmper from HEK-293 conditioned media (6 nM) were internalized similarly by MECs during the pulse. To deter- mine if Bmper internalizes into an endosomal transport pathway, we disrupted acidic endosomal transport during the chase phase of this experiment. Internalized Bmper was eliminated by MECs during the 2-h cold chase but, in striking contrast, Bmper levels were sustained in these cells in the presence of chloroquine. A ki- netic analysis indicated that the half-life of intracellular Bmper was 30 min and that steady-state levels were almost completely stabilized in cells treated with chloroquine (Fig. 2, b and c). These data provide further evidence of Bmper reentry from the extracel- lular space and indicate that Bmper is subsequently targeted for lysosomal degradation through an endocytic pathway. Bmp4 and Bmper are reciprocally regulated through internalization To test if other Bmp signaling components are also internalized and/or associated with Bmper-containing vesicles, we treated ENDOCYTIC REGULATION OF BMP SIGNALING BY BMPER • Kelley et al. 599