Biomedical subjects

D S Allison

Publications and source records attributed to D S Allison.

6 recordsLinked to original sources

PubMed1989

Mutations in the signal sequence of prepro-alpha-factor inhibit both translocation into the endoplasmic reticulum and processing by signal peptidase in yeast cells.

The effects of five single-amino-acid substitution mutations within the signal sequence of yeast prepro-alpha-factor were tested in yeast cells. After short pulse-labelings, virtually all of the alpha-factor precursor proteins from a wild-type gene were glycosylated and processed by signal peptidase. In contrast, the signal sequence mutations resulted in the accumulation of mostly unglycosylated prepro-alpha-factor after a short labeling interval, indicating a defect in translocation of the protein into the endoplasmic reticulum. Confirming this interpretation, unglycosylated mutant prepro-alpha-factor in cell extracts was sensitive to proteinase K and therefore in a cytosolic location. The signal sequence mutations reduced the rate of translocation into the endoplasmic reticulum by as much as 25-fold or more. In at least one case, mutant prepro-alpha-factor molecules were translocated almost entirely posttranslationally. Four of the five mutations also reduced the rate of proteolytic processing by signal peptidase in vivo, even though the signal peptide alterations are not located near the cleavage site. This study demonstrates that a single-amino-acid substitution mutation within a eucaryotic signal peptide can affect both translocation and proteolytic processing in vivo and may indicate that the recognition sequences for translocation and processing overlap within the signal peptide.

Acetylglucosaminidase↗

PubMed1988

Amphiphilicity is essential for mitochondrial presequence function.

We have shown earlier that a mitochondrial presequence peptide can form an amphiphilic helix. However, the importance of amphiphilicity for mitochondrial presequence function became doubtful when an artificial presequence, designed to be non-amphiphilic, proved to be active as a mitochondrial import signal. We now show experimentally that this 'non-amphiphilic' presequence peptide is, in fact, highly amphiphilic as measured by its ability to insert into phospholipid monolayers and to disrupt phospholipid vesicles. This result, and similar tests on three additional artificial presequences (two functionally active and one inactive), revealed that all active presequences were amphiphilic whereas the inactive presequence was non-amphiphilic. One of the active presequence peptides was non-helical in solution and in the presence of detergent micelles. We conclude that amphiphilicity is necessary for mitochondrial presequence function whereas a helical structure may not be essential.

Circular Dichroism↗

PubMed1988

Single-amino-acid substitutions within the signal sequence of yeast prepro-alpha-factor affect membrane translocation.

We used a genetic approach to identify point mutations in the signal sequence of a secreted eucaryotic protein, yeast alpha-factor. Signal sequence mutants were obtained by selecting for cells that partially mistargeted into mitochondria a fusion protein consisting of the alpha-factor signal sequence fused to the mature portion of an imported mitochondrial protein (Cox IV). The mutations resulted in replacement of a residue in the hydrophobic core of the signal sequence with either a hydrophilic amino acid or a proline. After reassembly into an intact alpha-factor gene, the substitutions were found to decrease up to 50-fold the rate of translocation of prepro-alpha-factor across microsomal membranes in vitro. Two of three mutants tested produced lower steady-state levels of alpha-factor in intact yeast cells, although the magnitude of the effect was less than that in the cell-free system.

Amino Acid Sequence↗

PubMed1987

Amino-terminal deletions in the presequence of an imported mitochondrial protein block the targeting function and proteolytic cleavage of the presequence at the carboxy terminus.

Subunit IV of yeast cytochrome oxidase is made in the cytosol with a 25-residue presequence. This presequence targets subunit IV into mitochondria and is removed by a protease in the matrix space. Here we show that removal of as few as 4 amino-terminal residues from the subunit IV presequence (which had been attached to the cytosolic protein dihydrofolate reductase) blocks import of the protein into mitochondria and proteolytic removal of the presequence by the soluble matrix protease. Thus, this protease requires not only an appropriate cleavage site at the carboxy-terminal end of the presequence, but also information at the extreme amino terminus of the presequence.

Amino Acid Sequence↗

PubMed1986

Artificial mitochondrial presequences.

Synthetic oligonucleotides were used to construct artificial mitochondrial presequences that contained, besides the initiator methionine, only arginine, serine, and leucine. The ratio of these three amino acids was adjusted to match that of basic, hydroxylated, and hydrophobic residues in natural mitochondrial presequences. When these sequences were fused to the N terminus of yeast cytochrome oxidase subunit IV lacking its own presequence, they directed the attached subunit IV to its correct intramitochondrial location in vivo. They also mediated import of subunit IV into isolated yeast mitochondria. In contrast, artificial sequences containing glutamine, arginine, and serine residues following the initiator methionine were inactive. Thus, the targeting function of mitochondrial presequences does not depend on specific amino acid sequences but may instead depend on the overall balance between basic, hydrophobic, and hydroxylated amino acids.

Amino Acid Sequence↗

PubMed1985

Effects of alterations in the 3' flanking sequence on in vivo and in vitro expression of the yeast SUP4-o tRNATyr gene.

The SUP4-o gene of Saccharomyces cerevisiae codes for an altered tRNATyr capable of suppressing ochre mutations. We constructed mutant SUP4-o genes with deletions in the 3'-flanking sequence and tested each for its ability to suppress ochre mutations in transformed yeast cells. The effects of the different 3' deletions on various aspects of in vitro transcription and RNA processing were also determined, using a yeast cell-free extract. Deletions that leave five or fewer consecutive T residues in the 3'-flanking sequence of SUP4-o were found to result in decreased efficiency of transcription termination, both in vitro and in vivo. Unexpectedly, the suppression strength of each mutant SUP4-o gene is highly correlated with the relative extent of transcription termination at the 3' end of the gene. This result indicates that SUP4-o readthrough transcripts are not efficiently processed to functional suppressor tRNA in yeast cells. Deletions that extend into the T cluster in the 3'-flanking sequence also significantly decrease the ability of SUP4-o to compete for a transcription factor that is limiting in our extracts. This latter finding implies that the 3'-flanking sequence of SUP4 plays a role in transcription factor binding.

Chromosome Deletion↗