Protein phosphorylation is essential for the regulation of cell growth, division, and differentiation in both prokaryotes and eukaryotes. Signal transduction in prokaryotes was previously thought to occur primarily by histidine kinases, involved in two-component signaling pathways. Lately, bacterial homologues of eukaryotictype serine/threonine kinases and phosphatases have been found to be necessary for cellular functions such as growth, differentiation, pathogenicity, and secondary metabolism. The Gram-positive bacteria Streptococcus agalactiae (group B streptococci, GBS) is an important human pathogen. We have identified and characterized a eukaryotic-type serine/threonine protein kinase (Stk1) and its cognate phosphatase (Stp1) in GBS. Biochemical assays revealed that Stk1 has kinase activity and localizes to the membrane and that Stp1 is a soluble protein with manganese-dependent phosphatase activity on Stk1. Mutations in these genes exhibited pleiotropic effects on growth, virulence, and cell segregation of GBS. Complementation of these mutations restored the wild type phenotype linking these genes to the regulation of various cellular processes in GBS. In vitro phosphorylation of cell extracts from wild type and mutant strains revealed that Stk1 is essential for phosphorylation of six GBS proteins. We have identified the predominant endogenous substrate of both Stk1 and Stp1 as a manganese-dependent inorganic pyrophosphatase (PpaC) by liquid chromatography/tandem mass spectrometry. These results suggest that these eukaryotictype enzymes regulate pyrophosphatase activity and other cellular functions of S. agalactiae.Protein phosphorylation is a principal mechanism of signal transduction governing various cellular processes such as physiology, growth, and development. Regulation of protein function or enzyme activity by covalent and reversible phosphorylation is crucial for the regulation of cellular responses of both prokaryotes and eukaryotes to dynamic internal and external environmental conditions. Signal transduction in prokaryotes was thought to occur primarily by histidine kinases that activate transcription by phosphorylation of cognate response regulators at aspartate residues (1). However, phosphorylation by serine kinases have also been described in prokaryotes. Examples of signal transduction by serine kinases, specifically in Gram-positives, include the well characterized and novel HPr system with bifunctional kinase/phosphatase activity necessary for carbon catabolite repression (for review, see Ref.2) and the isocitrate dehydrogenase kinase/phosphatase system (3, 4). Other examples of serine phosphorylation involves cognate pairs of kinases and phosphatases that regulate stress responses in Bacillus subtilis (5). Interestingly, although these serine kinases are homologous to the two-component histidine kinases (6), SpoIIE, which regulates sporulation in B. subtilis, is homologous to eukaryotic-type protein phosphatases (7).In eukaryotes, reversible protein phosphorylation via serine, threonin...

SummaryThe membrane integration of tail-anchored proteins at the endoplasmic reticulum (ER) is post-translational, with different tail-anchored proteins exploiting distinct cytosolic factors. For example, mammalian TRC40 has a well-defined role during delivery of tail-anchored proteins to the ER. Although its Saccharomyces cerevisiae equivalent, Get3, is known to function in concert with at least four other components, Get1, Get2, Get4 and Get5 (Mdy2), the role of additional mammalian proteins during tail-anchored protein biogenesis is unclear. To this end, we analysed the cytosolic binding partners of Sec61b, a well-defined substrate of TRC40, and identified Bat3 as a previously unknown interacting partner. Depletion of Bat3 inhibits the membrane integration of Sec61b, but not of a second, TRC40-independent, tail-anchored protein, cytochrome b5. Thus, Bat3 influences the in vitro membrane integration of tail-anchored proteins using the TRC40 pathway. When expressed in Saccharomyces cerevisiae lacking a functional GET pathway for tail-anchored protein biogenesis, Bat3 associates with the resulting cytosolic pool of non-targeted chains and diverts it to the nucleus. This Bat3-mediated mislocalisation is not dependent upon Sgt2, a recently identified component of the yeast GET pathway, and we propose that Bat3 either modulates the TRC40 pathway in higher eukaryotes or provides an alternative fate for newly synthesised tail-anchored proteins.

Tail-Anchored (TA) proteins are inserted into the endoplasmic reticulum (ER) membrane of yeast cells via the posttranslational Guided Entry of Tail-Anchored protein (GET) pathway. The key component of this targeting machinery is the ATPase Get3 that docks to the ER membrane by interacting with a receptor complex formed by the proteins Get1 and Get2. A conserved pathway is present in higher eukaryotes and is mediated by TRC40, homolog of Get3, and the recently identified membrane receptors WRB and CAML. Here, we used yeast lacking the GET1 and GET2 genes and substituted them with WRB and CAML. This rescued the growth phenotypes of the GET receptor mutant. We demonstrate that WRB and CAML efficiently recruit Get3 to the ER membrane and promote the targeting of the TA proteins in vivo. Our results show that the membrane spanning segments of CAML are essential to create a functional receptor with WRB and to ensure TA protein membrane insertion. Finally, we determined the binding parameters of TRC40 to the WRB/CAML receptor. We conclude that together, WRB and CAML are not only necessary but also sufficient to create a functional membrane receptor complex for TRC40. The yeast complementation assay can be used to further dissect the structure-function relationship of the WRB/CAML heteromultimer in the absence of endogenous receptor proteins.

Tail-anchored (TA) proteins are post-translationally inserted into membranes. The TRC40 pathway targets TA proteins to the endoplasmic reticulum via a receptor comprised of WRB and CAML. TRC40 pathway clients have been identified using in vitro assays, however, the relevance of the TRC40 pathway in vivo remains unknown. We followed the fate of TA proteins in two tissue-specific WRB knockout mouse models and found that their dependence on the TRC40 pathway in vitro did not predict their reaction to receptor depletion in vivo. The SNARE syntaxin 5 (Stx5) was extremely sensitive to disruption of the TRC40 pathway. Screening yeast TA proteins with mammalian homologues, we show that the particular sensitivity of Stx5 is conserved, possibly due to aggregation propensity of its cytoplasmic domain. We establish that Stx5 is an autophagy target that is inefficiently membrane-targeted by alternative pathways. Our results highlight an intimate relationship between the TRC40 pathway and cellular proteostasis.