IDENTIFICATION AND DNA SEQUENCE OF A NEW H+-ATPase IN THE UNICELLULAR GREEN ALGA CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE)

Insertional mutagenesis was used to identify genes involved in mating and/or zygote formation in the unicellular green alga Chlamydomonas reinhardtii Dangeard. Approximately 800 insertionally mutagenized transformants were examined, and a single nonagglutinating mutant was identified. Plasmid rescue was used to clone a genomic fragment containing transforming DNA. This fragment was then used to identify the wild-type copy of the gene disrupted during mutagenesis. The wild-type gene is transcribed during all stages of the life cycle and, based on sequence similarity, encodes a P2-type proton transporting ATPase. The gene is referred to as Pmh1 for plasma membrane H ⁺ -ATPase. PMH1 displays the greatest sequence similarity to ATPases from two parasitic flagellates and a raphidophytic alga but not to the ATPase from a closely related green alga. We propose that PMH1 represents a distinct H ⁺ -ATPase isoform expressed in flagellates.     

Efficient uptake of Yersinia pseudotuberculosis via integrin receptors involves a Rac1–Arp 2/3 pathway that bypasses N-WASP function

Efficient uptake of Yersinia pseudotuberculosis into cultured mammalian cells is the result of high-affinity binding of invasin to beta1 chain integrins. We demonstrate here that uptake requires Rac1 and Arp 2/3 function. Bacterial uptake was stimulated by GTPgammaS, but was inhibited in mammalian cells transfected with the interfering Rac1-N17 derivative. Rac1 was found to be activated in response to integrin engagement by invasin, whereas Rac1 and Arp 2/3 were found to be intensely localized around phagosomes bearing bacteria, indicating a specific role for Rac1 signalling from the nascent phagosome to downstream effectors. To determine whether the Arp 2/3 complex was a component of this proposed pathway, cells overproducing various derivatives of Scar1/WAVE1, an Arp 2/3-binding protein, were analysed. Sequestration of Arp 2/3 away from the phagocytic cup as a result of Scar1/WAVE1 overproduction dramatically inhibited uptake. To determine whether signalling from Rac1 to Arp 2/3 occurred via N-WASP, uptake was analysed in a cell line lacking expression of WASP and N-WASP. Uptake was unaffected by the absence of these proteins, indicating that beta1 integrin signalling from Rac1 to Arp 2/3 can occur in the absence of N-WASP function.