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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.

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Isolation of SOI1. (A) KRY18-1A (MATα kex2Δ) and  JBY11-r1 (MATα kex2Δ soi1-2), expressing Y713A Kex2p under  the control of the GAL1 promoter from plasmid pCWKX21 (55),  were transformed with either pBS32 (vector control) or pSOI1.1,  and were analyzed for their Soi and Vps phenotypes, as described  in Materials and Methods. For the mating assay, strains were  shifted to glucose for 6 h before testing mating competence. (B)  Restriction map of pSOI1.1 and analysis of subcloned fragments.  At the top is shown a restriction map of the insert from pSOI1.1.  Indicated above the map is the extent and direction of the SOI1  open reading frame. Restriction fragments from pSOI1.1 were  subcloned into either pBS32 or pRS313, transformed into JBY96  (MATa/MATα, soi1-2/soi1-2), and tested for complementation of  the Spo− phenotype (scores are shown to the right of each subclone). Restriction sites are indicated as follows: C, ClaI; P, PstI;  N, NcoI; S, SalI; X, XhoI. The ClaI site indicated by an asterisk is  not unique, but is the only one within the insert not blocked by  dam methylation.
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Figure 2: Isolation of SOI1. (A) KRY18-1A (MATα kex2Δ) and JBY11-r1 (MATα kex2Δ soi1-2), expressing Y713A Kex2p under the control of the GAL1 promoter from plasmid pCWKX21 (55), were transformed with either pBS32 (vector control) or pSOI1.1, and were analyzed for their Soi and Vps phenotypes, as described in Materials and Methods. For the mating assay, strains were shifted to glucose for 6 h before testing mating competence. (B) Restriction map of pSOI1.1 and analysis of subcloned fragments. At the top is shown a restriction map of the insert from pSOI1.1. Indicated above the map is the extent and direction of the SOI1 open reading frame. Restriction fragments from pSOI1.1 were subcloned into either pBS32 or pRS313, transformed into JBY96 (MATa/MATα, soi1-2/soi1-2), and tested for complementation of the Spo− phenotype (scores are shown to the right of each subclone). Restriction sites are indicated as follows: C, ClaI; P, PstI; N, NcoI; S, SalI; X, XhoI. The ClaI site indicated by an asterisk is not unique, but is the only one within the insert not blocked by dam methylation.

Mentions: In addition to Soi+ and Vps− phenotypes, soi1 mutations also resulted in a severe sporulation defect. As measured by the production of viable spores (38), sporulation efficiency was reduced ∼2,500-fold in soi1 homozygous diploid strains (data not shown). However, asci were not identifiable by microscopic inspection of cultures of soi1 homozygous diploids. Because of the stringent nature of the sporulation phenotype, we exploited it to clone SOI1 by complementation, as described in Materials and Methods. Screening a single-copy yeast genomic library, only 2 of 20,000 transformants were Spo+. Plasmid loss and retransformation experiments confirmed that complementation of this phenotype was plasmid dependent (data not shown). These two Spo+ transformants contained the same plasmid, hereafter called pSOI1.1, that had a 12-kb insert. Transformation of a MATα soi1-2 kex2Δ strain with pSOI1.1 complemented both the Vps− and Soi+ phenotypes caused by the soi1-2 mutation (Fig. 2 A).


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)

Isolation of SOI1. (A) KRY18-1A (MATα kex2Δ) and  JBY11-r1 (MATα kex2Δ soi1-2), expressing Y713A Kex2p under  the control of the GAL1 promoter from plasmid pCWKX21 (55),  were transformed with either pBS32 (vector control) or pSOI1.1,  and were analyzed for their Soi and Vps phenotypes, as described  in Materials and Methods. For the mating assay, strains were  shifted to glucose for 6 h before testing mating competence. (B)  Restriction map of pSOI1.1 and analysis of subcloned fragments.  At the top is shown a restriction map of the insert from pSOI1.1.  Indicated above the map is the extent and direction of the SOI1  open reading frame. Restriction fragments from pSOI1.1 were  subcloned into either pBS32 or pRS313, transformed into JBY96  (MATa/MATα, soi1-2/soi1-2), and tested for complementation of  the Spo− phenotype (scores are shown to the right of each subclone). Restriction sites are indicated as follows: C, ClaI; P, PstI;  N, NcoI; S, SalI; X, XhoI. The ClaI site indicated by an asterisk is  not unique, but is the only one within the insert not blocked by  dam methylation.
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Related In: Results  -  Collection

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Figure 2: Isolation of SOI1. (A) KRY18-1A (MATα kex2Δ) and JBY11-r1 (MATα kex2Δ soi1-2), expressing Y713A Kex2p under the control of the GAL1 promoter from plasmid pCWKX21 (55), were transformed with either pBS32 (vector control) or pSOI1.1, and were analyzed for their Soi and Vps phenotypes, as described in Materials and Methods. For the mating assay, strains were shifted to glucose for 6 h before testing mating competence. (B) Restriction map of pSOI1.1 and analysis of subcloned fragments. At the top is shown a restriction map of the insert from pSOI1.1. Indicated above the map is the extent and direction of the SOI1 open reading frame. Restriction fragments from pSOI1.1 were subcloned into either pBS32 or pRS313, transformed into JBY96 (MATa/MATα, soi1-2/soi1-2), and tested for complementation of the Spo− phenotype (scores are shown to the right of each subclone). Restriction sites are indicated as follows: C, ClaI; P, PstI; N, NcoI; S, SalI; X, XhoI. The ClaI site indicated by an asterisk is not unique, but is the only one within the insert not blocked by dam methylation.
Mentions: In addition to Soi+ and Vps− phenotypes, soi1 mutations also resulted in a severe sporulation defect. As measured by the production of viable spores (38), sporulation efficiency was reduced ∼2,500-fold in soi1 homozygous diploid strains (data not shown). However, asci were not identifiable by microscopic inspection of cultures of soi1 homozygous diploids. Because of the stringent nature of the sporulation phenotype, we exploited it to clone SOI1 by complementation, as described in Materials and Methods. Screening a single-copy yeast genomic library, only 2 of 20,000 transformants were Spo+. Plasmid loss and retransformation experiments confirmed that complementation of this phenotype was plasmid dependent (data not shown). These two Spo+ transformants contained the same plasmid, hereafter called pSOI1.1, that had a 12-kb insert. Transformation of a MATα soi1-2 kex2Δ strain with pSOI1.1 complemented both the Vps− and Soi+ phenotypes caused by the soi1-2 mutation (Fig. 2 A).

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