Purification and Light-Dependent Phosphorylation of a Candidate Fusion Protein, the Photoreceptor Cell Peripherin/rds

Abstract

The proteins peripherin/rds and rom-1 form a protein complex in the rims of photoreceptor outer segment disk membranes. Peripherin/rds plays an essential role in the morphogenesis and maintenance of disk membrane structure, with peripherin/rds gene mutations resulting in photoreceptor cell degeneration. We report two different chromatographic procedures for the purification of native peripherin/rds from bovine photoreceptor cell outer segments and show that the protein is a phosphoprotein that promotes membrane fusion in vitro. During one procedure, peripherin/rds was copurified in association with rom-1 by hyroxylapatite and Mono Q FPLC. During the other, it was purified free from rom-1 by concanavalin-A affinity chromatography and chromatofocusing. Analysis of homogeneous peripherin/rds from the second procedure showed that exposure of photoreceptor outer segments to light resulted in the incorporation of nearly 2 mol of phosphate per mole of peripherin/rds and a concomitant shift in the isoelectric point of the protein. In addition, we found that recombination of purified peripherin/rds into lipid vesicles increased membrane fusion, with more rapid fusion detected with phosphorylated peripherin/rds. In conclusion, studies with purified peripherin/rds reveal that the protein undergoes light-dependent phosphorylation and that it may function in membrane fusion.

Phosphorylation of the Ca2+-Binding Protein CaBP4 by Protein Kinase C ζ in Photoreceptors

“…Thus, elucidating the factors controlling CaBP4 activity may yield insights into the underlying pathology of CSNB2. Light-dependent phosphorylation regulates the physiological activity of several key players in the phototransduction cascade, including rhodopsin, opsin, phosducin, RGS-9 (regulator of G-protein signaling 9), centrins, ␤-subunit of the insulin receptor, abLIM (actin binding LIM protein family), peripherin, and phosphodiesterase (Kuhn and Dreyer, 1972;Lee et al, 1984;Boesze-Battaglia et al, 1997;Roof et al, 1997;Hayashi et al, 2000;Hu et al, 2001;Rajala et al, 2002;Trojan et al, 2003). Phosphorylation of Ca 2ϩ -binding proteins, including CaM, can affect their Ca 2ϩ -binding capability and interaction with target proteins (Benaim and Villalobo, 2002).…”

ROM-1 potentiates photoreceptor specific membrane fusion processes

“…ROM-1 was purified using a strategy originally developed for the purification of P/rds that relied on a combination of Concanavalin-A Sepharose affinity chromatography and chromatofocusing (Boesze-Battaglia, Kong et al 1997). Briefly, ROS disk membranes were prepared from frozen, dark-adapted retinas (J. Lawson Inc.) using Ficoll flotation (Smith, Stubbs et al 1975).…”

“…ROM-1 was separated from P/rds by chromatofocusing as described in detail (BoeszeBattaglia, Kong et al 1997). Mono Q HR 5/5 columns, PBE-94 and Polybuffer were purchased from Pharmacia.…”

“…The plasma membrane forms vesicles as the OS is broken and reseals (Boesze-Battaglia, Albert et al 1992).When the plasma membrane reseals it forms both inside out and outside out vesicles (Boesze-Battaglia 1997). The orientation reflects the extra cellular plasma membrane surface or the intra-diskal surface Vesicles enriched in P/rds and rom-1 were prepared essentially as described previously and are designated rim specific vesicles (RSV) (Boesze-Battaglia, Kong et al 1997). Rim specific vesicles (RSV's) were prepared from the dialyzed lipid rich fraction of ROS-disk Con A chromatography by freeze/thawing as described above.…”

“…Previous studies in our laboratory have suggested that disks at the apical tip of the OS fuse spontaneously to delineate a packet of disks (Boesze-Battaglia 1997) and that the tetraspanin protein, peripherin/rds (P/rds a.k.a. Prph, Rds and peripherin-2) participates in this membrane fusion process in vitro (Boesze-Battaglia 1997;Boesze-Battaglia, Kong et al 1997;Boesze-Battaglia, Lamba et al 1998). Deletion of a region including the amphiphilic fusion peptide domain of P/rds in transgenic Xenous laevis resulted in the mis-localization of the mutated P/rds (Tam, Moritz et al 2002;Tam, Moritz et al 2004), supporting a key functional role for this domain.…”