Mammalian cells express two VPS4 proteins both of which are involved in intracellular protein trafficking

The yeast Vps4 protein (Vps4p) is a member of the AAA protein family (ATPas es associated with diverse cellular activities) and a key player in the tra nsport of proteins out of a prevacuolar endosomal compartment. In human cel ls, we identified two non-allelic orthologous proteins (VPS4-A and VPS4-B) of yeast Vps4p. The human VPS4-A and VPS4-B proteins display a high degree of sequence identity to each other (80 %) and to the yeast Vps4 protein (59 and 60 %, respectively). Yeast cells lacking a functional VPS4 gene exhibi t a temperature-sensitive growth defect and mislocalise a carboxypeptidase Y-invertase fusion protein to the cell surface. Heterologous expression of human VPS4 genes in vps4 mutant yeast strains led, in the case of human VPS 4-A, to a partial and, in the case of human VPS4-B, to a complete suppressi on of the temperature-sensitive growth defect. The vacuolar protein sorting defect of vps4 mutant yeast cells was complemented completely by heterolog ous expressed human VPS4-B protein, and partially by the human VPS4-A prote in. Expression of mutant human VPS4-A (E228Q) and VPS4-B (E235Q) proteins, harbouring single amino acid exchanges in their AAA domains, induced domina nt-negative vacuolar protein sorting defects in wild-type yeast cells in bo th cases. Two-hybrid experiments suggest that the human VPS4-A and VPS4-B p roteins can form heteromeric complexes, and subcellular localisation experi ments indicate that both human VPS4 proteins associate with endosomal compa rtments in yeast. Based on these results, we conclude that both human VPS4 proteins are involved in intracellular protein trafficking, presumably at a late endosomal protein transport step, similar to the Vps4p in yeast. (C) 2001 Academic Press.