Translocation of nascent secretory proteins across membranes can occur late in translation.

Signal recognition particle (SRP) causes an arrest in the translation of nascent secretory proteins in a wheat germ cell-free system. In order to examine at what point during the synthesis of a secretory protein its translocation across the endoplasmic reticulum (ER) membrane can occur, SRP was used to arrest nascent chain elongation at various times during a synchronous translation, thus allowing the generation of nascent chains of increasing length. It was found that SRP can still bring about an arrest as late as when an average of two-thirds of nascent IgG light chain was completed. Rough microsomes were added to translations blocked with SRP to determine if such relatively long nascent chains could still be translocated across the membrane. It was found that nascent chains which had been arrested by SRP, regardless of their length, could be translocated into rough microsomes. In the case of IgG light chain, translocation levels of 50% were still observed with nascent chains corresponding to as much as 70-75% of the intact preprotein. Similar results were observed for the nascent bovine prolactin precursor. These results demonstrate that the synthesis of secretory proteins can be uncoupled from their translocation, and that fairly large nascent chains are capable of crossing the membrane of the ER post-translationally.

Mechanisms of cytoskeletal regulation: functional and antigenic diversity in human erythrocyte and brain beta spectrin.

A study of human erythrocyte and brain spectrin with particular emphasis on the beta subunits revealed a structural homology but functional dissimilarity between these two molecules. Six monoclonal antibodies raised to human erythrocyte beta spectrin identify three of the four proteolytically defined domains of erythrocyte beta spectrin. Five of these monoclonal antibodies cross-react with human brain spectrin. None of a previously identified set of alpha erythrocyte spectrin monoclonal antibodies [Yurchenco et al: J Biol Chem 257:9102, 1982] reacted with brain spectrin. A domain map generated by limited tryptic digestion shows that brain spectrin is composed of proteolytically resistant domains analogous to erythrocyte spectrin, but the brain protein is more basic. The binding of brain spectrin to erythrocyte ankyrin, both in solution and on erythrocyte IOVs, yielded an association constant approximately 100 time weaker than for erythrocyte spectrin. The binding of azido-calmodulin under native conditions was specific for the erythrocyte beta subunit but was not calcium dependent. In contrast, azido-calmodulin bound only to the alpha subunit of brain spectrin in a calcium-dependent manner. The similarity of structure but modified functional characteristics of the brain and erythrocyte beta spectrins suggest that these proteins serve different cellular roles.

Mechanism of cytoskeletal regulation (I): functional differences correlate with antigenic dissimilarity in human brain and erythrocyte spectrin.

Human erythrocyte and brain spectrin (fodrin, calspectin) have been compared quantitatively with respect to the extent and sites of antigenic and functional similarity. Brain spectrin cross-reacts strongly with approx. 1% of the epitopes in erythrocyte spectrin, but weakly with at least 50%. The distribution of shared determinants is not uniform. Brain spectrin is most deficient in epitopes characteristic of the 80 kDa and 52 kDa domains of the alpha-subunit (alpha-I and alpha-III) and of terminal portions of the 28 kDa and 74 kDa domains of the beta-subunit (beta-I and beta-IV). The functions associated with these domains also differ between the two proteins. Brain spectrin does not undergo extensive polymerization and binds calmodulin at a different site. The unique ability of erythrocyte spectrin to oligomerize beyond the tetramer reflects its role in the membrane skeleton. Non-erythroid spectrins probably function as specific linkers between membrane receptors and the filamentous cytoskeleton. In this sense, they may act as regulated transducers of information flow between the membrane and the cytoplasmic matrix.