Bsp1p, a Possible β-Adducin Homolog, Is Required for Vps13p-Dependent TGN Homotypic Fusion

Yeast Vps13p is a cytosolic factor required for multiple vesicular transport/fusion events, including trans-Golgi network (TGN)–late endosome and late endosome-Golgi transport, TGN homotypic fusion and spore membrane maturation. Humans express four VPS13 homologs (VPS13A-D) and loss of function mutations in these genes have been associated with distinct neurodegenerative or neurodevelopmental diseases. Chorea Acanthocytosis (ChAc) is an autosomal recessive Huntington's-like neurodegenerative disease caused by VPS13A null mutations. In addition to loss of striatal neurons, ChAc patients exhibit aberrantly shaped red blood cells (RBCs), acanthocytes, suggesting defects in the actin cytoskeleton. Recent experiments showed that, in RBCs, human VPS13A is in a complex with β-actin and β-adducin, suggesting a role in actin organization. Bioinformatic analysis identified a possible β-adducin homolog in yeast, Bsp1p, an actin-binding protein associated with cytokinesis. Here, we have found that extracts from bsp1 mutant yeast cells are defective for in vitro TGN homotypic fusion. Bsp1p was purified from yeast as a soluble TAP-tagged protein. TGN homotypic fusion assay involves the fusion of Ste13-HA (SHA) membranes with membranes containing Kex2p for Kex2p-mediated cleavage that separates the HA epitope tag from the DPAP (Ste13) domain. Cleaved HA was quantified by measuring the fraction of DPAP activity in the supernatant that could not be immunoprecipitated using anti-HA antibody. In bsp1-deletion extracts, ~10% of SHA is processed by Kex2p-mediated cleavage over the course of 20 minutes. In bsp1+ membranes, the percentage of SHA processed increases from ~10% at 0 minutes to 25% at 20 minutes. Purified Bsp1p restored TGN homotypic fusion activity to mutant extracts, suggesting that Bsp1p is directly required for this membrane fusion event. These results support the hypothesis that Bsp1p is a yeast homolog of β-adducin and connect Vps13p function to the organellar actin cytoskeleton. Sequence alignments of fungal Bsp1p and metazoan β-adducin homologs provide further support for this conclusion. Future studies will focus on how Bsp1p is recruited to membranes and whether Bsp1p and Vps13p function in complex with actin. Understanding the function of yeast Vps13p will further clarify the fundamental defects behind human VPS13 diseases.