Prenylation of proteins by the intact lens.
OBJECTIVE
To describe and identify proteins prenylated by the intact rat lens.


METHODS
Lenses from young rats were incubated for 24 hours in TC199 medium containing 22 microM lovastatin and 110 microCi/ml [3H]mevalonolactone. Proteins of the epithelium and fiber cells were separated by high-performance liquid chromatography (HPLC) and one- and two-dimensional electrophoresis, and the 3H label was detected by fluorography. Treatment of labeled proteins with methyl iodide released isoprenes that were identified by HPLC analysis. The identity of the prenylated proteins was probed by N-terminal sequence analysis and matrix-assisted, laser desorption ionization-mass spectrometry (MALDI-MS).


RESULTS
The pattern of protein prenylation by epithelial and fiber cells was similar. Most of the labeled proteins were water insoluble and were assumed to be membrane associated. A group of 21-kDa to 29-kDa proteins were most intensely labeled and were modified mainly with geranylgeranyl. A highly labeled, approximately 80-kDa insoluble protein and a lesser labeled, 64-kDa soluble protein were the only other significant prenylated proteins. Both were farnesylated. MALDI-MS analysis suggested that the 80-kDa protein is a cytokeratin. N-terminal sequence analysis indicated that the 64-kDa soluble protein is beta-tubulin.


CONCLUSIONS
A limited set of proteins are prenylated by the young rat lens. The 21-kDa to 29-kDa proteins were modified mainly by geranylgeranyl and are likely members of the numerous small GTP binding proteins. The authors express caution about accepting the identities of the 64-kDa and 80-kDa proteins as beta-tubulin and cytokeratin-1, respectively--proteins in these families do not contain the required CAAX motif. The 80-kDa farnesylated protein could be novel and unique to the lens, because no farnesylated protein of this size has been previously reported.
Cenedella
R J
RJ
Department of Biochemistry, Kirksville College of Osteopathic Medicine, Missouri 63501, USA.
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003459:Crystallins / Q000032:analysis; D015180:Electrophoresis, Gel, Two-Dimensional; D007633:Keratins / Q000032:analysis; D007908:Lens, Crystalline / Q000737:chemistry / Q000378:metabolism*; D013058:Mass Spectrometry; D008970:Molecular Weight; D009924:Organ Culture Techniques; D017368:Protein Prenylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D014404:Tubulin / Q000032:analysis
D003459:Crystallins; D014404:Tubulin; D007633:Keratins