The biogenesis of endothelial-specific Weibel-Palade bodies (WPB) is poorly understood, despite their key role in both haemostasis and inflammation. Biogenesis of specialized organelles of haemopoietic cells is often adaptor protein complex 3-dependent (AP-3-dependent), and AP-3 has previously been shown to play a role in the trafficking of both WPB membrane proteins, P-selectin and CD63. However, WPB are thought to form at the trans Golgi network (TGN), which is inconsistent with a role for AP-3, which operates in post-Golgi trafficking. We have therefore investigated in detail the mechanisms of delivery of these two membrane proteins to WPB. We find that P-selectin is recruited to forming WPB in the transGolgi by AP-3-independent mechanisms that use sorting information within both the cytoplasmic tail and the lumenal domain of the receptor. In contrast, CD63 is recruited to already-budded WPB by an AP-3-dependent route. These different mechanisms of recruitment lead to the presence of distinct immature and mature populations of WPB in human umbilical vein endothelial cells (HUVEC). Weibel-Palade bodies (WPB) are endothelial-specific cigarshaped secretory organelles (1, reviewed in 2) which store the haemostatic protein von Willebrand factor (VWF) (3) within a limiting membrane that includes the leukocyte receptor P-selectin (4,5) and the tetraspanin CD63 (6). Their composition and tissue distribution thus place them at the centre of both haemostasis and inflammation. Despite this, WPB biogenesis is not well understood. The little that is known suggests that they share at least some characteristics with lysosome-related organelles (LRO).LRO are cell-type-specific structures with secretory functions, including melanosomes, cytotoxic T-lymphocyte lytic granules, platelet dense granules, the MHC class II compartment, basophil and azurophil granules, and lamellar bodies. They are thus often seen in cells with a haemopoietic lineage. All share some characteristics with endosomes and lysosomes (and some are called secretory lysosomes), including protein composition, low intraorganellar pH and access from the endocytic pathway without re-entering the biosynthetic pathway at the level of the Golgi (7-10). Their biogenesis is often marked by the delivery of specialized components to endocytic organelles, thereby transforming them into LRO. One common LRO characteristic is a role for the adaptor protein complex 3 (AP-3) in this process. In endothelial cells, AP-3 has been reported as being required for P-selectin recruitment to WPB (11); it is also reported to affect the trafficking of CD63 in NRK and NIH3T3 cells (12).However, we also know that WPB formation is driven by VWF (13), whose heterologous expression in HEK293 cells can cause the appearance of cigar-shaped, ultrastructurally correct 'WPB' that recruit appropriate membrane proteins and respond to secretagogue (14). Moreover, WPB are thought to form at the trans Golgi network (TGN), and heterologously expressed P-selectin is recruited to forming secretory granul...

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The biogenesis of secretory lysosomes, which combine characteristics of both lysosomes and secretory granules, is currently of high interest. In particular, it is not clear whether delivery of membrane proteins to the secretory lysosome requires lysosomal, secretory granule, or some novel targeting determinants. Heterologous expression of P-selectin has established that this membrane protein contains targeting signals for both secretory granules and lysosomes. P-selectin is therefore an ideal probe with which to determine the signals required for targeting to secretory lysosomes. We have exploited subcellular fractionation and immunofluorescence microscopy to monitor targeting of transiently expressed wild-type and mutant horseradish peroxidase (HRP)-P-selectin chimeras to secretory lysosomes of Rbl-2H3 cells. The exposure of the HRP chimeras to intracellular proteolysis was also determined as a third monitor of secretory lysosome targeting. Our data show that HRP-P-selectin accumulates in secretory lysosomes of Rbl-2H3 cells using those cytoplasmic sequences previously found to be sufficient for targeting to conventional lysosomes. This work highlights the similar sorting signals used for targeting of membrane proteins to conventional lysosomes and secretory lysosomes.