Over-expression of protein kinase C-α enhances platelet-derived growth factor- and phorbol ester- but not calcium ionophore-induced formation of prostaglandins in NIH 3T3 fibroblasts

AbstractOver-expression of human protein kinase C-α in murine NIH 3T3 fibroblasts is associated with an increased platelet-derived growth factor- and phorbol ester-mediated formation of prostaglandins, whereas the calcium ionophore-induced release of arachidonic acid metabolites is unaffected; however, the differences of arachidonic acid and prostaglandin formation are much more pronounced with platelet-derived growth factor than with phorbol ester. Platelet-derived growth factor induces an identical elevation of intracellular free calcium in control and protein kinase C-α over-expressing cells; the phorbol ester has no effect on intracellular free calcium in both cell lines. These results demonstrate that protein kinase C-α may couple to arachidonic acid cascade in NIH 3T3 fibroblasts

Cotranslational intersection between the SRP and GET targeting pathways to the ER of Saccharomyces cerevisiae.

Targeting of transmembrane proteins to the endoplasmic reticulum proceeds via either the SRP or the GET pathway, consisting of Get1-5 and Sgt2. While SRP cotranslationally targets membrane proteins containing one or multiple transmembrane domains, the GET pathway posttranslationally targets proteins containing a single, C-terminal transmembrane domain termed tail-anchor. Here we dissect the role of the SRP and GET pathway in the sorting of homologous, two-membrane spanning K(+) channel proteins termed Kcv, Kesv, and Kesv-VV. We show that Kcv was targeted to the ER cotranslationally via its N-terminal transmembrane domain, while Kesv-VV was targeted posttranslationally via its C-terminal transmembrane domain, which recruited Get4-5/Sgt2 and Get3. Unexpectedly, nascent Kcv did not only recruit SRP, but also the Get4-5 module of the GET pathway to ribosomes. Ribosome-binding of Get4-5 was independent of Sgt2 and was strongly outcompeted by SRP. The combined data indicate a previously unrecognized cotranslational interplay between the SRP and GET pathways

Interaction of the cotranslational Hsp70 Ssb with ribosomal proteins and rRNA depends on its lid domain

Cotranslational chaperones assist in de novo folding of nascent polypeptides in all organisms. In yeast, the heterodimeric ribosome-associated complex (RAC) forms a unique chaperone triad with the Hsp70 homologue Ssb. We report the X-ray structure of full length Ssb in the ATP-bound open conformation at 2.6 Å resolution and identify a positively charged region in the α-helical lid domain (SBDα), which is present in all members of the Ssb-subfamily of Hsp70s. Mutational analysis demonstrates that this region is strictly required for ribosome binding. Crosslinking shows that Ssb binds close to the tunnel exit via contacts with both, ribosomal proteins and rRNA, and that specific contacts can be correlated with switching between the open (ATP-bound) and closed (ADP-bound) conformation. Taken together, our data reveal how Ssb dynamics on the ribosome allows for the efficient interaction with nascent chains upon RAC-mediated activation of ATP hydrolysis.This work was supported by the Deutsche Forschungsgemeinschaft (DFG) through FOR967 (to I.S. and S.R.), GRK1188 and the Leibniz programme (to I.S.), SFB 746 (to S.R.), and RO 1028/5-1 (to S.R.), and by HBIGS fellowships (to G.V.G. and F.A.W.), and by the Excellence Initiative of the German federal and state governments (BIOSS-2) (to S.R). I.S. is an investigator of the Cluster of Excellence: CellNetworks