Makino Lab Members
Maria Solovyeva - Laboratory Specialist
Xiao-Hong Wen - Postdoctoral Fellow
Xiao-Hong Wen Research Interests: What is the function of palmitoylation on rhodopsin? Rhodopsin is a G-protein coupled receptor that captures light with an 11-cis retinal chromophore for transduction into an electrical response. Rhodopsin features two conserved and consecutive cysteine sites on its carboxyl-terminal tail, which undergo palmitoylation (Figure 1). Two 16-carbon palmitates attach post-translationally and
	Figure 1. Two-dimensional model of a wild-type mouse rhodopsin. Cysteine residues at positions 322 and 323 (red) were mutated to non-palmitoylatable threonine and serine residues, preventing the attachment of palmitates at the C-terminal end of the eighth a-helix. The C-terminal serine (334, 338, 343) and threonine (336, 340, 342) residues are light-dependent phosphorylation sites (blue).
anchor the fourth cytoplasmic loop into the disk membrane. The palmitoylation sites are located near regions responsible for G-protein activation and phosphorylation sites necessary for rhodopsin deactivation. Palmitoylation might therefore impact the activity of rhodopsin. Furthermore, the reversible nature of palmitoylation suggests that it might participate in light adaptation. In order to clarify the role of palmitoylation, the two cysteine attachment sites on rhodopsin were replaced by a threonine and a serine in knock-in mice by Drs. Wang and Lem at the New England Medical Center. The two mutated sites were neither phosphorylated nor palmitoylated. We used a suction electrode to record the electrical responses of rods with the palmitoylation-deficient rhodopsin to flashes. The rising phase of the photocurrent response was unaffected but the shutoff was faster and sensitivity was reduced (Figure 2). Biochemical assays indicated that light-exposed, palmitoylation-deficient rhodopsins became phosphorylated at a faster rate. Thus, palmitoylation modulates rhodopsin activity by slowing the rate of rhodopsin phosphorylation.
	Figure 2. Normalized averaged responses of mutant (Palm-/-, red traces) and WT (black traces) rods to bright and dim flashes at 500 nm. The Palm-/- response recovered earlier than that of WT. In addition the Palm-/- responded to subsaturating flashes was slightly smaller.
Tomoki Isayama - Instructor
Dmitriy Alexeev - Instructor
Quanhua He - Postdoctoral Fellow
Yajie Wang - Postdoctoral Fellow