BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 248, 469–472 (1998) ARTICLE NO. RC989007 Control of Rab5 and Rab7 Expression by the Isoprenoid Pathway Chiara Laezza,* Cecilia Bucci,* Mariarosaria Santillo,† Carmelo B. Bruni,* and Maurizio Bifulco* , ‡ ,1 *Centro di Endocrinologia ed Oncologia Sperimentale ‘‘G. Salvatore’’ del CNR and Dipartimento di Biologia e Patologia Cellulare e Molecolare ‘‘L. Califano’’ and †Dipartimento di Neuroscienze e della Comunicazione Interumana, Universita ` di Napoli ‘‘Federico II’’; and ‡Dipartimento di Medicina Sperimentale e Clinica ‘‘G. Salvatore,’’ Universita ` di Catanzaro, Italy Received June 16, 1998 anylgeranyltransferase type I (GGTase-I) and gera- Rab proteins are small molecular mass GTP-ases in- nylgeranyltransferase type II (GGTase-II) (5, 6). The volved in the regulation of vescicular transport. The isoprenoid substrates required for prenylation are all ability of rab proteins to carry out their role in intra- derived from mevalonate (MVA), the product of the re- cellular membrane traffic requires the post-transla- action catalyzed by 3-hydroxy-3-methylglutaryl coen- tional attachment to their C-terminus of a geranylger- zyme A (HMG-CoA) reductase. HMG-CoA reductase is anyl group, an isoprenoid lipid moiety derived from under the feedback control of cholesterol and can be mevalonate. Here we report that depletion of intracel- competitively inhibited by lovastatin, a fungal drug (7- lular mevalonate by lovastatin in FRTL-5 thyroid cells 9). Lovastatin has been widely utilized to define the specifically resulted in a four-fold increase of Rab5 and role of prenylation in the targeting of some prenylated Rab7 protein levels. This increase was reversed within proteins to the membranes. Thus, treatment of cells 4 h upon addition of mevalonate. Similarly lovastatin with lovastatin results in the accumulation of small also induced, at same extent, mRNA levels. Lovastatin GTP-ases in the cytosol, as they do not undergo post- effect was not common to other prenylated proteins. translational modification due to decline in the level of Moreover incubation with cycloheximide abolished endogenous isoprenyl groups. However the functional the observed increase in lovastatin treated cells, sug- gesting that the effect is mediated by newly synthe- differences between processed and unprocessed pro- sized protein. These findings demonstrate that Rab5 teins cannot be fully accounted for by differences in and Rab7 expression are regulated by the isoprenoid membrane association since the prenylated C-terminus pathway.  1998 Academic Press of some of these proteins is also directly involved in protein-protein interaction (10). Apart from these ef- fects, lovastatin also causes a significant increase in the cellular levels of HMG-CoA reductase and has also Prenylation is a post-translational modification re- been shown to arrest cells in culture in the G1 phase quired to direct a protein to the cellular site where it of the cell division cycle (7, 11). In spite of the important can perform its biological function. Several members role played by Rab proteins in the regulation of endo- of the broad family of low molecular mass GTP-binding and exocitic pathway up to now no data are available proteins in mammalian cells are modified by 15-carbon on the regulation of Rab expression. The current inves- farnesyl or 20-carbon geranylgeranyl isoprenoids linked tigation was undertaken to determine the effect of lo- to carboxyl-terminal cysteine residues (1-3). In particu- vastatin on expression of Rab proteins. In this paper lar Rab proteins are modified by geranylgeranylation. we demonstrated that addition of lovastatin to FTRL- These proteins are localized to distinct intracellular 5 cells specifically increased the amount of Rab5 and compartments along the endocytic and exocytic path- Rab7 proteins and mRNA. This implies a regulation way, and regulate specific steps of vesicular membrane of the expression of these proteins by the isoprenoid traffic (4). Prenylation of these proteins is cata- pathway. lyzed by two different enzymes designated protein ger- MATERIALS AND METHODS 1 To whom correspondence should be addressed at: Dip. Biol. Pat. Cell. Mol., Univ. ‘‘Federico II,’’ via S. Pansini, 5 80131 Napoli, Italy. Materials. Lovastatin was a kind gift of Dr. A.W. Alberts of the Merck, Sharp and Dohme Co.. Mevalonic acid lactone, cycloheximide, Fax: 39-81-7701016. E-mail: maubiful@unina.it. 0006-291X/98 $25.00 Copyright  1998 by Academic Press All rights of reproduction in any form reserved. 469