Peripherin-2 (also known as peripherin/rds), a photoreceptor specific tetraspanin protein, is required to maintain normal cell structure through its role in renewal processes requiring membrane fusion. It is the first tetraspanin fusogen and has been shown to directly mediate fusion between disk membranes and opposing membranes to maintain the highly ordered structure of rod outer segments. Localized to the C terminus of human, bovine, and murine peripherin-2 is an amphiphilic fusion peptide domain (residues 312-326) and a highly conserved region upstream of this domain that we hypothesize is essential for fusogenic function. Our previous studies indicated that substitution of a threonine for a proline at position 296 within this highly conserved region enhanced fusion activity. In this study we wanted to determine whether this proline is essential with the introduction of three additional substitutions of proline with alanine, leucine, and glutamic acid. Wild type, P296T, P296A, P296L, and P296E mutants of peripherin-2 were expressed as His 6-tagged full-length proteins in Madin-Darby canine kidney (MDCK) cells. All of the proteins were localized to intracellular membranes and detected as 42-kDa monomers by Western blot analysis. The wild type, P296A, and P296L assembled into core tetramers; in contrast the P296T and P296E formed higher order oligomers. Fusogenic activity of full-length protein expressed in MDCK membranes and purified protein reconstituted in model membrane liposomes was determined using fluorescence quenching techniques. Fusion activity was decreased in the P296L, P296A, and P296E mutants both in endogenous MDCK membranes and in model liposomes. Collectively, these results suggest that the proline at position 296 is necessary for optimal function. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.