Limits...
SOI1 encodes a novel, conserved protein that promotes TGN-endosomal cycling of Kex2p and other membrane proteins by modulating the function of two TGN localization signals.

Brickner JH, Fuller RS - J. Cell Biol. (1997)

Bottom Line: Deletion of SOI1 improved retention of Tyr713Ala Kex2p in the pro-alpha-factor processing compartment but, unlike the original soi1 alleles, did not increase the half-life of Tyr713Ala Kex2p.This hypothesis was confirmed in several ways.TLS2 delayed exit of Kex2p from the TGN, whereas TLS1 did not affect this step.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor 48109-0606, USA.

ABSTRACT
Localization of yeast Kex2 protease to the TGN requires a signal (TLS1) in its cytosolic tail (C-tail). Mutation of TLS1 results in rapid transit of Kex2p to the vacuole. Isolation of suppressors of the Tyr713Ala mutation in TLS1 previously identified three SOI genes. SOI1, cloned by complementation of a sporulation defect, encodes a novel, hydrophilic 3,144-residue protein with homologues in Caenorhabditis elegans, Drosophila melanogaster, and humans. Epitope-tagged Soi1p existed in a detergent-insensitive, sedimentable form. Deletion of SOI1 impaired TGN localization of wild-type Kex2p and a fusion protein containing the C-tail of Ste13p, and also caused missorting of carboxypeptidase Y and accelerated vacuolar degradation of the Vps10p sorting receptor. Deletion of SOI1 improved retention of Tyr713Ala Kex2p in the pro-alpha-factor processing compartment but, unlike the original soi1 alleles, did not increase the half-life of Tyr713Ala Kex2p. These results suggested that Soi1p functions at two steps in the cycling of Kex2p and other proteins between the TGN and prevacuolar compartment (PVC). This hypothesis was confirmed in several ways. Soi1p was shown to be required for optimal function of TLS1. Suppression of the Tyr713Ala mutation by mutation of SOI1 was shown to be caused by activation of a second signal (TLS2) in the Kex2p C-tail. TLS2 delayed exit of Kex2p from the TGN, whereas TLS1 did not affect this step. We propose that Soi1p promotes cycling of TGN membrane proteins between the TGN and PVC by antagonizing a TGN retention signal (TLS2) and facilitating the function of a retrieval signal (TLS1) that acts at the PVC.

Show MeSH

Related in: MedlinePlus

Sequence of Soi1p and analysis of conserved sequences. (A) The deduced amino acid sequence of Soi1p (GenBank/EMBL/ DDBJ accession number AF001317). (B) Schematic representation of Soi1p and the deduced amino acid sequence from C. elegans open  reading frame T08G11.1. Percent identity and similarity (using a PAM250 substitution matrix) were calculated using Clustal W (49) and  plotted for segments of 120 amino acids (the COOH-terminal segment was 87 amino acids). Upper plot, percent identity; lower plot, percent similarity. A second potential C. elegans homologue of ∼3,200 codons (not shown) can be formed by joining two adjacent, predicted open reading frames from cosmid C25H3 (sequences C25H3.8 and C25H3.9, GenBank/EMBL/DDBJ accession No. U29535).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139830&req=5

Figure 3: Sequence of Soi1p and analysis of conserved sequences. (A) The deduced amino acid sequence of Soi1p (GenBank/EMBL/ DDBJ accession number AF001317). (B) Schematic representation of Soi1p and the deduced amino acid sequence from C. elegans open reading frame T08G11.1. Percent identity and similarity (using a PAM250 substitution matrix) were calculated using Clustal W (49) and plotted for segments of 120 amino acids (the COOH-terminal segment was 87 amino acids). Upper plot, percent identity; lower plot, percent similarity. A second potential C. elegans homologue of ∼3,200 codons (not shown) can be formed by joining two adjacent, predicted open reading frames from cosmid C25H3 (sequences C25H3.8 and C25H3.9, GenBank/EMBL/DDBJ accession No. U29535).

Mentions: Genetic linkage between soi1-2 and the pSOI1.1 insert confirmed that pSOI1.1 contained the SOI1 gene (see Materials and Methods). Efforts to isolate smaller complementing fragments from pSOI1.1 were unsuccessful, suggesting that the length of the complementing gene was between 6 and 10 kb (Fig. 2 B). Sequencing of the insert from pSOI1.1 revealed a single long open reading frame of 9,432 bp (GenBank accession number AF001317; Fig. 2 B) predicted to encode a 3,144-residue polypeptide of 358 kD (Fig. 3 A). This open reading frame was flanked by fragments of the UBI4 and SDH2 genes, and comparison of this sequence to that from this region of chromosome XII (GenBank/EMBL/DDBJ accession number Z73145) revealed no significant differences. BLAST and FASTA similarity searches of the GenBank/EMBL/DDBJ database using the deduced amino acid sequence of Soi1p identified an open reading frame from Caenorhabditis elegans with significant similarity to SOI1 (T08G11.1; GenBank/EMBL/DDBJ accession number 1546759). This open reading frame would encode a protein of 3,212 amino acids that is 22% identical and 42% similar to Soi1p over its entire length, with the highest degree of similarity in the NH2 and COOH termini (Fig. 3 B). Given the strong conservation of NH2- and COOH-terminal domains along with nearly identical overall length and similar sequence composition, T08G11.1 represents a likely Soi1p homologue in C. elegans, suggesting conservation of the protein and its function between yeast and metazoans.


SOI1 encodes a novel, conserved protein that promotes TGN-endosomal cycling of Kex2p and other membrane proteins by modulating the function of two TGN localization signals.

Brickner JH, Fuller RS - J. Cell Biol. (1997)

Sequence of Soi1p and analysis of conserved sequences. (A) The deduced amino acid sequence of Soi1p (GenBank/EMBL/ DDBJ accession number AF001317). (B) Schematic representation of Soi1p and the deduced amino acid sequence from C. elegans open  reading frame T08G11.1. Percent identity and similarity (using a PAM250 substitution matrix) were calculated using Clustal W (49) and  plotted for segments of 120 amino acids (the COOH-terminal segment was 87 amino acids). Upper plot, percent identity; lower plot, percent similarity. A second potential C. elegans homologue of ∼3,200 codons (not shown) can be formed by joining two adjacent, predicted open reading frames from cosmid C25H3 (sequences C25H3.8 and C25H3.9, GenBank/EMBL/DDBJ accession No. U29535).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139830&req=5

Figure 3: Sequence of Soi1p and analysis of conserved sequences. (A) The deduced amino acid sequence of Soi1p (GenBank/EMBL/ DDBJ accession number AF001317). (B) Schematic representation of Soi1p and the deduced amino acid sequence from C. elegans open reading frame T08G11.1. Percent identity and similarity (using a PAM250 substitution matrix) were calculated using Clustal W (49) and plotted for segments of 120 amino acids (the COOH-terminal segment was 87 amino acids). Upper plot, percent identity; lower plot, percent similarity. A second potential C. elegans homologue of ∼3,200 codons (not shown) can be formed by joining two adjacent, predicted open reading frames from cosmid C25H3 (sequences C25H3.8 and C25H3.9, GenBank/EMBL/DDBJ accession No. U29535).
Mentions: Genetic linkage between soi1-2 and the pSOI1.1 insert confirmed that pSOI1.1 contained the SOI1 gene (see Materials and Methods). Efforts to isolate smaller complementing fragments from pSOI1.1 were unsuccessful, suggesting that the length of the complementing gene was between 6 and 10 kb (Fig. 2 B). Sequencing of the insert from pSOI1.1 revealed a single long open reading frame of 9,432 bp (GenBank accession number AF001317; Fig. 2 B) predicted to encode a 3,144-residue polypeptide of 358 kD (Fig. 3 A). This open reading frame was flanked by fragments of the UBI4 and SDH2 genes, and comparison of this sequence to that from this region of chromosome XII (GenBank/EMBL/DDBJ accession number Z73145) revealed no significant differences. BLAST and FASTA similarity searches of the GenBank/EMBL/DDBJ database using the deduced amino acid sequence of Soi1p identified an open reading frame from Caenorhabditis elegans with significant similarity to SOI1 (T08G11.1; GenBank/EMBL/DDBJ accession number 1546759). This open reading frame would encode a protein of 3,212 amino acids that is 22% identical and 42% similar to Soi1p over its entire length, with the highest degree of similarity in the NH2 and COOH termini (Fig. 3 B). Given the strong conservation of NH2- and COOH-terminal domains along with nearly identical overall length and similar sequence composition, T08G11.1 represents a likely Soi1p homologue in C. elegans, suggesting conservation of the protein and its function between yeast and metazoans.

Bottom Line: Deletion of SOI1 improved retention of Tyr713Ala Kex2p in the pro-alpha-factor processing compartment but, unlike the original soi1 alleles, did not increase the half-life of Tyr713Ala Kex2p.This hypothesis was confirmed in several ways.TLS2 delayed exit of Kex2p from the TGN, whereas TLS1 did not affect this step.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor 48109-0606, USA.

ABSTRACT
Localization of yeast Kex2 protease to the TGN requires a signal (TLS1) in its cytosolic tail (C-tail). Mutation of TLS1 results in rapid transit of Kex2p to the vacuole. Isolation of suppressors of the Tyr713Ala mutation in TLS1 previously identified three SOI genes. SOI1, cloned by complementation of a sporulation defect, encodes a novel, hydrophilic 3,144-residue protein with homologues in Caenorhabditis elegans, Drosophila melanogaster, and humans. Epitope-tagged Soi1p existed in a detergent-insensitive, sedimentable form. Deletion of SOI1 impaired TGN localization of wild-type Kex2p and a fusion protein containing the C-tail of Ste13p, and also caused missorting of carboxypeptidase Y and accelerated vacuolar degradation of the Vps10p sorting receptor. Deletion of SOI1 improved retention of Tyr713Ala Kex2p in the pro-alpha-factor processing compartment but, unlike the original soi1 alleles, did not increase the half-life of Tyr713Ala Kex2p. These results suggested that Soi1p functions at two steps in the cycling of Kex2p and other proteins between the TGN and prevacuolar compartment (PVC). This hypothesis was confirmed in several ways. Soi1p was shown to be required for optimal function of TLS1. Suppression of the Tyr713Ala mutation by mutation of SOI1 was shown to be caused by activation of a second signal (TLS2) in the Kex2p C-tail. TLS2 delayed exit of Kex2p from the TGN, whereas TLS1 did not affect this step. We propose that Soi1p promotes cycling of TGN membrane proteins between the TGN and PVC by antagonizing a TGN retention signal (TLS2) and facilitating the function of a retrieval signal (TLS1) that acts at the PVC.

Show MeSH
Related in: MedlinePlus