LMA1 Binds to Vacuoles at Sec18p (NSF), Transfers upon ATP Hydrolysis to a t-SNARE (Vam3p) Complex, and Is Released during Fusion

AbstractVacuole fusion requires Sec18p (NSF), Sec17p (α-SNAP), Ypt7p (GTP binding protein), Vam3p (t-SNARE), Nyv1p (v-SNARE), and LMA1 (l ow Mr a ctivity 1, a heterodimer of thioredoxin and IB2). LMA1 requires Sec18p for saturable, high-affinity binding to vacuoles, and Sec18p “priming” ATPase requires both Sec17p and LMA1. Either the sec18-1 mutation and deletion of IB2, or deletion of both IB2 and p13 (an IB2 homolog) causes a striking synthetic vacuole fragmentation phenotype. Upon Sec18p ATP hydrolysis, LMA1 transfers to (and stabilizes) a Vam3p complex. LMA1 is released from vacuoles in a phosphatase-regulated reaction. This LMA1 cycle explains how priming by Sec18p is coupled to t-SNARE stabilization and to fusion

I2B is a Small Cytosolic Protein that Participates in Vacuole Fusion

Saccharomyces cerevisiae vacuole inheritance requires two low molecular weight activities, LMA1 and LMA2. LMA1 is a heterodimer of thioredoxin and protease B inhibitor 2 (IB2). Here we show that the second low molecular weight activity (LMA2) is monomeric IB2. Though LMA2 / IB2 was initially identified as a protease B inhibitor, this protease inhibitor activity is not related to its ability to promote vacuole fusion: ( i ) Low M r protease B inhibitors cannot substitute for LMA1 or LMA2, ( ii ) LMA1 and LMA2 promote the fusionof vacuoles from a strain that has no protease B, ( iii ) low concentrations of LMA2 that fully inhibit protease B do not promote vacuole fusion, and ( iv ) LMA1, in which is complexed with thioredoxin,is far more active than LMA2 / IB2 in promoting vacuole fusion and far less active in inhibiting protease B. These studies establish a new function for IB2

Vacuole acidification is required for trans-SNARE pairing, LMA1 release, and homotypic fusion

ABSTRACT Vacuole fusion occurs in three stages: priming, in which Sec18p mediates Sec17p release, LMA1 (low M r activity 1) relocation, and cis-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex disassembly; docking, mediated by Ypt7p and trans-SNARE association; and fusion of docked vacuoles. Ca 2؉ and calmodulin regulate late stages of the reaction. We now show that the vacuole proton gradient, generated by the vacuolar proton ATPase, is needed for trans-SNARE complex formation during docking and hence for the subsequent LMA1 release. Though neither the vacuolar Pmc1p Ca 2؉ -ATPase nor the Vcx1p Ca 2؉ ͞H ؉ exchanger are needed for the fusion reaction, they participate in Ca 2؉ and ⌬ H ؉ homeostasis. Fusion itself does not require the maintenance of trans-SNARE pairs

Vacuole acidification is required for trans-SNARE pairing, LMA1 release, and homotypic fusion

Vacuole fusion occurs in three stages: priming, in which Sec18p mediates Sec17p release, LMA1 (low M(r) activity 1) relocation, and cis-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex disassembly; docking, mediated by Ypt7p and trans-SNARE association; and fusion of docked vacuoles. Ca(2+) and calmodulin regulate late stages of the reaction. We now show that the vacuole proton gradient, generated by the vacuolar proton ATPase, is needed for trans-SNARE complex formation during docking and hence for the subsequent LMA1 release. Though neither the vacuolar Pmc1p Ca(2+)-ATPase nor the Vcx1p Ca(2+)/H(+) exchanger are needed for the fusion reaction, they participate in Ca(2+) and Δμ(H)(+) homeostasis. Fusion itself does not require the maintenance of trans-SNARE pairs

A novel chromatography system to isolate active ribosomes from pathogenic bacteria

We have developed a novel chromatography for the rapid isolation of active ribosomes from bacteria without the use of harsh conditions or lengthy procedures that damage ribosomes. Ribosomes interact with an alkyl linker attached to the resin, apparently through their RNA component. Examples are given with ribosomes from Escherichia coli, Deinococcus radiodurans, and with clinical isolates of Streptococcus pneumoniae and methicillin-resistant Staphylococcus aureus (MRSA). The ribosomes obtained by this method are unusually intact, so that highly active ribosomes can now be isolated from the clinical isolates, enabling significantly improved in vitro functional assays that will greatly assist the discovery and development of new ribosomally targeted antibiotics