Seven genes in Saccharomyces cerevisiae are predicted to code for membrane-spanning proteins (designated AVT1-7) that are related to the neuronal ␥-aminobutyric acid-glycine vesicular transporters. We have now demonstrated that four of these proteins mediate amino acid transport in vacuoles. One protein, AVT1, is required for the vacuolar uptake of large neutral amino acids including tyrosine, glutamine, asparagine, isoleucine, and leucine. Three proteins, AVT3, AVT4, and AVT6, are involved in amino acid efflux from the vacuole and, as such, are the first to be shown directly to transport compounds from the lumen of an acidic intracellular organelle. This function is consistent with the role of the vacuole in protein degradation, whereby accumulated amino acids are exported to the cytosol. Protein AVT6 is responsible for the efflux of aspartate and glutamate, an activity that would account for their exclusion from vacuoles in vivo. Transport by AVT1 and AVT6 requires ATP for function and is abolished in the presence of nigericin, indicating that the same pH gradient can drive amino acid transport in opposing directions. Efflux of tyrosine and other large neutral amino acids by the two closely related proteins, AVT3 and AVT4, is similar in terms of substrate specificity to transport system h described in mammalian lysosomes and melanosomes. These findings suggest that yeast AVT transporter function has been conserved to control amino acid flux in vacuolar-like organelles.Caenorhabditis elegans UNC-47 (1) and the vertebrate homologues from rat (VGAT, 1) and mouse (VIAAT, 2) are synaptic vesicular transporters that are expressed exclusively in inhibitory neurons (see also Ref. 3) and are specific for the neurotransmitters ␥-aminobutyric acid (GABA) 1 and glycine. These proteins differ in sequence, structure, and bioenergetics from the previously characterized family of vesicular transporters that package monoamines and acetylcholine (for current reviews, see Refs. 4 and 5). Common to all of these transporters is the fact that the movement of substrate from the cytosol into synaptic vesicles is driven by a proton electrochemical gradient that is generated by the action of a vacuolar-type H ϩ -ATPase and involves the exchange of lumenal protons.A search for other vertebrate proteins related to the GABAglycine vesicular transporters has led recently to the isolation and expression of cDNAs that, surprisingly, code for Na ϩ
Most genetic elements that employ reverse transcription generate a terminally redundant genomic RNA that serves as the template for this reaction. Because the identical polyadenylation signal is present in each terminally redundant segment, synthesis of this RNA requires that this signal be ignored on the first pass of the transcription machinery, then recognized and used on the second pass. We have studied the mechanism of this differential poly(A) site use in one family of retroid elements, the hepatitis B viruses (hepadnaviruses). Our results indicate that two features are involved: the presence of a variant poly(A) signal (TATAAA) and the participation of multiple sequences 5' to this signal that act to increase the efficiency of its use. Deletion of these upstream elements abolishes proper poly(A) site use, despite the presence of the poly(A) signal and downstream GT-and T-rich motifs known to be required for polyadenylation. Sequences from the corresponding regions of retroviral genomes can restore proper processing to these hepadnaviral deletion mutants. Thus, functionally analogous upstream elements exist in other classes of retroid elements, including those employing the canonical AATAAA hexanucleotide signal.
The nucleotide sequences of two different cDNAs, CEHS48 and CEHS41, coding for the 16,000 dalton heat shock proteins (hsps) of Caenorhabditis elegans have been determined. CEHS48 codes for a polypeptide of 135 amino acids, approximately 15 fewer than the complete protein while CEHS41 is missing approximately 46 amino acids. From nucleotide 113 to the TAA termination signal the extent of homology between the sequences is 91%. Toward the 5' ends, the homology drops to 20% and results in completely divergent amino acid sequences. The 3' noncoding regions are only 30% homologous. Only CEHS48 contains a poly(A) signal and a poly(A) tail, suggesting that CEHS41 has an incomplete 3' end. The region from amino acid 43 to amino acid 115 shows extensive homology with corresponding regions in the four small hsps of Drosophila melanogaster and in mammalian alpha-crystallin. Two-dimensional gel analysis of in vitro synthesized hsp16 reveals the existence of five distinct components of identical molecular weights, but with different isoelectric points.
Hepatitis B viruses replicate by reverse transcription of a genomic RNA which harbors terminal redundancies. The synthesis of this RNA requires that transcription proceed twice through the polyadenylation (pA) site which, in mammalian strains, is flanked by the variant hexanucleotide UAUAAA and a T-rich downstream domain. These core elements are by themselves virtually defective in 3' end processing and require multiple upstream accessory elements which regulate pA site use. In ground squirrel hepatitis B virus (GSHV), one of these signals (PS1; -215 to -107 relative to UAUAAA) is transcribed only at the 3' end of genomic RNA and as such is analogous to retroviral U3 sequences. PS1 cooperates with other signals to enhance pA site use to very high levels and can be further sub-divided into two regions (A and B) which contribute equally to 3' end processing. Critical residues within PS1B have been localized to a 15 bp A/T-rich stretch which displays homology to other known upstream activating signals. A 15 bp segment within PS1A which has the identical A/T content but a divergent primary sequence plays a diminished role in processing. Furthermore, PS1 can activate GSHV core element usage autonomously. This stimulation has been shown to be additive since multiple copies of PS1 progressively increase polyadenylation, a phenomenon which also demands that PS1 exert its influence from a variety of distances from the hexanucleotide signal.
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