Green fluorescent protein (GFP) from Aequorea victoria was used as an in vivo reporter protein when fused to the carboxy-terminus of the Pho84 phosphate permease of Saccharomyces cerevisiae. Both components of the fusion protein displayed their native functions and revealed a cellular localization and degradation of the Pho84-GFP chimera consistent with the behavior of the wild-type Pho84 protein. The GFP-tagged chimera allowed for a detection of conditions under which the Pho84 transporter is localized to its functional environment, i.e. the plasma membrane, and conditions linked to relocation of the protein to the vacuole for degradation. By use of the methodology described, GFP should be useful in studies of localization and degradation also of other membrane proteins in vivo.z 1999 Federation of European Biochemical Societies.

No abstract

The PHO84 and PHO89 genes of Saccharomyces cerevisiae encode two high-affinity phosphate cotransporters of the plasma membrane. Hydropathy analysis suggests a secondary structure arrangements of the proteins in 12 transmembrane domains. The derepressible Pho84 and Pho89 transporters appear to have characteristic similarities with the phosphate transporters of Neurospora crassa. The Pho84 protein catalyzes a proton-coupled phosphate transport at acidic pH, while the Pho89 protein catalyzes a sodium-dependent phosphate uptake at alkaline pH. The Pho84 transporter can be stably overproduced in the cytoplasmic membrane of Escherichia coli, purified and reconstituted in a functional state into proteoliposomes.

NatB is one of three major N-terminal acetyltransferase (NAT) complexes (NatA-NatC), which co-translationally acetylate the N-termini of eukaryotic proteins. Its substrates account for about 21% of the human proteome, including well known proteins such as actin, tropomyosin, CDK2, and αsynuclein (aSyn). Human NatB (hNatB) mediated N-terminal acetylation of αSyn has been demonstrated to play key roles in Parkinson's disease pathogenesis and as a potential therapeutic target for hepatocellular carcinoma.Here we report the cryo-EM structure of hNatB bound to a CoA-aSyn conjugate, together with structure-guided analysis of mutational effects on catalysis. This analysis reveals functionally important differences with human NatA and Candida albicans NatB, resolves key hNatB protein determinants for aSyn Nterminal acetylation, and identifies important residues for substrate-specific recognition and acetylation by NatB enzymes. These studies have implications for developing small molecule NatB probes and for understanding the mode of substrate selection by NAT enzymes.