Besides its ability to inhibit the core apoptotic machinery, BclxL has been shown to modulate a number of other aspects of cellular physiology. Its overexpression, for instance, has been correlated with high tumour grade and increased ability to invade and metastasize, independently of its ability to sustain survival in the absence of matrix attachment. Lomitapide Mesylate Bcl-xL has been found to complement S.cerevisiae genes that facilitate the switch from glycolytic to oxidative metabolism. Bcl-xL is also able to modulate calcium homeostasis, stimulate synapse formation, slow cell cycle progression, modulate autophagy, increase mitochondrial fission/fusion and modulate metabolite exchange across the outer mitochondrial membrane. Some of these ”unconventional” Bcl-xL activities could be explained by its ability to interact with proteins other than the pro-apoptotic ”BH3 only” factors. Bcl-xL has indeed been shown to interact with VDAC1, with the IP3 Receptor, with Beclin1 and a number of other proteins. Bcl-xL is both a cytosolic and a membrane-associated protein. While cytosolic Bcl-xL appears to be a homodimer, the quaternary structure of membrane-bound Bcl-xL has not been investigated in details, although it has been reported that it could be engaged in high molecular weight complexes. In the present study we present evidence that Bcl-xL is indeed part of high molecular weight complexes and we attempt to carry a comprehensive analysis of proteins able to bind Bcl-xL using Tandem Affinity Purification. Interestingly, we found that Bcl-xL interacts with proteins involved in several cellular processes. Among them, the secretory pathway was one of the most represented pathways. With the aim of validating the list of proteins found to interact with Bcl-xL, we decided to focus our analysis on the interaction between Bcl-xL and Praf2, a small protein belonging to the Prenylated Rab Acceptor family, with a predicted role in ER to Golgi transport. We show that Praf2 induces apoptotic cell death upon expression and that Bcl-xL counteracts Praf2’s apoptotic activity. Finally, we show that Praf2 silencing in U2OS cells makes them more resistant to apoptosis induced by the cytotoxic drug etoposide. Bcl-xL is a C-tail anchored protein with the ability to localise on several intracellular membranes. In order to have a wide picture of the set of membrane proteins interacting with Bcl-xL, we performed Tandem Affinity Purification from total cellular membranes of HeLa cells stably expressing TAP-tagged Bcl-xL. Given that nonionic detergents like Triton-100 or NP-40 are known to alter the conformation of proteins of the Bcl-2 family, all the experiments were performed in presence of CHAPS, a detergent that leaves the conformation of Bcl-xL unchanged. As listed in Table 1, we found many proteins co-purifying with TAPBcl-xL. However, those are likely to reflect true protein-protein interactions for at least two reasons. First of all, as shown in lane 1 of Fig. 2C, purifications from the same cell line carrying a TAPtag-only construct recover almost undetectable proteins, suggesting that all recovered proteins are indeed associated to Bcl-xL. Second, as discussed below, using this procedure we found coeluting with Bcl-xL many proteins known to be able to specifically interact with Bcl-xL. We have sub-divided the list of proteins found to interact with Bcl-xL into the functional Butenafine hydrochloride categories discussed below. It is worth to notice that all the proteins found are either trans-membrane proteins or soluble proteins localised in intracellular organelles. Most of them localise to the sub-cellular compartments known to be targeted by Bcl-xL. Many of them are multiple components of protein complexes. These observations constitute a good indication of the validity of the approach used and of the specificity and sensitivity of the technique used. We can therefore draw the conclusion that Bcl-xL can be engaged in several different protein complexes at several sub-cellular sites.