Abstract
G protein gamma-subunits are isoprenylated and carboxyl-methylated at the C-terminal cysteine, which is indispensable for the function of photoreceptor G protein transducin (T alpha beta gamma). However, the physiological role of the methylation and its reversibility have been unclear. Here we isolated methylated and non-methylated forms of farnesylated T beta gamma, and demonstrated that the methylation remarkably facilitates not only the membrane association of T beta gamma but also the subunit interaction between T alpha and T beta gamma. Consequently, the functional coupling of transducin with light-activated receptor, metarhodopsin II, was stabilized by the methylation, resulting in acceleration of GTP gamma S (guanosine 5'-3-O-(thio) triphosphate) binding to T alpha. An examination of the reversibility of the methylation suggested that T gamma is kept fully methylated in rod outer segments. These observations indicate that the methylation of T gamma plays an important role in the most efficient photon-signal transduction process in rod cells.
Highlights
Weisolated methylated and non-methylated formwse found that it totally impairs thefunction of the py-subunit of farnesylated TPy, and demonstrated that the methyl- (Tpy) which is indispensable for the GDP/GTP exchange reacation remarkably facilitatesnotonlythe membrane tion onTa catalyzed by light-activated receptor, metarhodopsin association of TPy but alsothesubunitinteraction
Geranylgeranylationof y-subunit of brain G protein leads to a tight membrane association of the py-subunit (10S)i.milarly, the bindingof p2lraS to the plasma membrane requires the C-terminal farnesyl group (111, which is essential for expression of the transforming activity [12]
Since the methylationof Ty, unlike farnesylation, ispossibly a reversible process [18], it isimportant toknow whether the methylatioonf isoprenylated cysteine residue alters theG protein function
Summary
Later than Ty with a low yield of recovery. Measurements ofMembraneAssociation ofTP?cStripped ROS mem-. Bis 10 m MOPS-NaOH (pH 7.5), 2 m MgC12, 1 m D m , 0.1 m ated strippedROS membranes in thperesence or absence of purified Ta phenylmethylsulfonylfluoride, pg/ml leupeptin, and50kallikrein (final concentration, 1.0 p ~ in) 1.08 ml of buffer B supplemented with inhibitor unitdml aprotinin.Buffer C is 100 m Tris-HC1(pH 7.5) and 100 m NaCI This mixture was incubated at 4 "C for 5 min, and . Ta bound to thecolumn was eluted withbuffer B branes were purified from the retinasby sucrose stepwise density gracontaining 600 m NaCI, and loaded onto a gel filtration column, dient [20] and washed twice with the ROS buffer. 4 "C to precipitate microsomal membranes, which were subsequently washed five times with buffer A (hypotonic buffer) to remove soluble
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