Polytopic Membrane Topogenesis

We have had a long-standing interest in understanding the mechanism whereby polytopic membrane proteins are targeted, integrated and translocated into the cell membrane. Several years ago a number of point mutations in the rhodopsin molecule were found in patients with retinitis pigmentosa (RP). These mutations frequently involve the substitution of charged for uncharged amino acids or uncharged for charged amino acids in various parts of rhodopsin. A number of unrelated studies on other proteins had already demonstrated the importance of charged amino acids in the regulation of membrane insertion and protein trafficking through the cell. Improperly processed or integrated opsin could lead to photoreceptor dysfunction and/or degradation leading to accumulation of cellular "debris" or increased inflammatory cell activity related to the removal of this debris.

We have been studying the asymetric insertion of polytopic membrane proteins using bovine rhodospin and the NCX as model proteins. Through these studies we have learned that there are multiple internal topogenic signal sequences in molecules such as rhodopsin (Friedlander and Blobel, 1985). Using site directed mutagenesis and other molecular biological approaches, we have been able to identify those regions of the rhodopsin molecule important for its normal insertion into the cell membrane (Audigier, Friedlander and Blobel, 1987). We have recently developed an improved in vitro coupled transcription/translocation system and used it to demonstrate that efficient targeting and translocation of nascent opsin, like tail-anchored proteins, is not dependent upon protein synthesis. (Kanner, Friedlander and Simon, 2002). However, in contrast to the tail-anchored proteins, the targeting and translocation of the amino terminus is efficient only while the nascent opsin is still functionally attached to its biosynthetic ribosome. This targeting and translocation occurs efficiently only with short nascent opsin polypeptides. The in vitro efficiency achieved with this assay is very high, suggesting that it mimics the in vivo reaction pathway. Furthermore, there is strong correlation between the efficiencies of the post- and co-translational reactions. Unlike the translocation of the tail-anchored proteins, SRP is required for both co-translational and post-translational targeting of nascent opsin to the ER membrane. We have most recently used this system to demonstrate that post-translational targeting and translocation requires nucleotide triphosphates but not cytosolic proteins (Kanner, et al, 2002). The addition of GTP alone was sufficient to fully restore targeting. The addition of ATP was not specifically required and non-hydrolysable analogs of ATP that blocked 90% of the ATPase activity also had no inhibitory effect on translocation. Finally, we have conducted a series of studies with NCXs from the heart and eye that differ in that while both have N-terminal signal sequences the requirements for cleavage differ between the two tissues. We have found that neither the presence nor cleavage of the N-terminal signal sequence in the cardiac NCX is required for membrane assembly of a functional exchanger (Sahin-Toth, et al 1995). In contrast, the first 65aa of the photoreceptor NCX constitute an uncleaved signal sequence required for the efficient membrane targeting and proper membrane integration of the exchanger (McKiernan and Friedlander, 1998).

Relevant Publications:

Roos, K.P., Jordan, M.C., Fishbein, M.C., Ritter, M.R., Friedlander, M., Chang, H.C., Rahgozar, P., Han, T., Garcia, A., MacClellan, W.R., Ross, R.S., and K.D. Philipson. (2007). Hypertrophy and Heart Failure in Mice Overexpressing the Cardiac Sodium-Calcium Exchanger. Journal of Cardiac Failure 13:318-329.

Kanner, E., Friedlander, M. and Simon, S.M. (2003). Co-translational targeting and translocation of the amino terminus of opsin across the ER membrane requires GTP but not ATP. J. Biol. Chem. 278:7920-7926.

Kanner, E., Klein, I.K., Friedlander, M., and Simon, S.M. (2002). The amino terminus of opsin translocates "post-translationally" as efficiently as co-translationally. Biochemistry 41:7707-7715.

McKiernan, C.J. and Friedlander, M. (1999). The retinal rod Na+/Ca2+,K+ exchanger contains a non-cleaved signal sequence required for translocation of the N-terminus. J. Biol. Chem. 274:38177-38182.

Sahin-Toth, M., Kaback, R., Friedlander, M. (1996). Association between the amino and carboxy terminal halves of lactose permease is specific and mediated by multiple transmembrane domains. Biochemistry, 35:2016-2021.

Sahin-Toth, M., Nicoll, D.A., Frank, J., Philipson, K., Friedlander, M. (1995). The cleaved N-terminal signal sequence of the cardiac sodium-calcium exchanger is not required for functional membrane integration. Biochem. Biophys. Res. Comm. 212:968-974.

Audigier, Y., M. Friedlander and G. Blobel (1989). Multiple topogenic sequences in bovine opsin. Proc. Natl. Acad. Sci. (U.S.A.) 84:5783-5787.

Mostov, K.E., Friedlander M., and G. Blobel (1986). Structure and function of the receptor for polymeric immunoglobulins. Biochem Soc Symp 51:113-5 .

Friedlander, M. and G. Blobel (1985). Bovine opsin has more than one signal sequence. Nature 318:338-343.

Mostov, K.M., M. Friedlander and G. Blobel (1984). The receptor for transepithelial transport of IgA and IgM contains multiple Ig-like domains. Nature 308:37-43.