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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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Deletion of SOI1 reveals a second TLS in the C-tail of  Kex2p. (A) BFY106-4D (SOI1 kex2Δ) and JBY134.1 (soi1Δ-1  kex2Δ) expressing WT Kex2p, Y713A Kex2p or I718tail Kex2p,  under the control of the GAL1 promoter, were shifted from galactose to glucose for the indicated times before testing mating  competence. (B and D) JBY135-1A (soi1Δ-1) and JBY135-2D  (SOI1) carried CEN plasmids expressing either I718tail Kex2p  (pCWKX10-I718tail; B) or C-tailΔ Kex2p (pCWKX17; D) from  the KEX2 promoter (34, 55). (C) JBY173 (soi1Δ-2 pep4 prb1  prc1) and CB018 (SOI1 pep4 prb1 prc1) carried pCWKX10-I718tail. Strains were labeled for 10 min (B and C) or 5 min (D),  chased, and samples were collected at the indicated times after  the addition of chase. Cells were lysed, and Kex2p was immunoprecipitated using antiserum raised against the Kex2p lumenal  domain (55). WT Kex2p is indicated by closed arrows. I718tail  Kex2p (B and C) and C-tailΔ Kex2p (D) are indicated by open  arrows.
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Figure 7: Deletion of SOI1 reveals a second TLS in the C-tail of Kex2p. (A) BFY106-4D (SOI1 kex2Δ) and JBY134.1 (soi1Δ-1 kex2Δ) expressing WT Kex2p, Y713A Kex2p or I718tail Kex2p, under the control of the GAL1 promoter, were shifted from galactose to glucose for the indicated times before testing mating competence. (B and D) JBY135-1A (soi1Δ-1) and JBY135-2D (SOI1) carried CEN plasmids expressing either I718tail Kex2p (pCWKX10-I718tail; B) or C-tailΔ Kex2p (pCWKX17; D) from the KEX2 promoter (34, 55). (C) JBY173 (soi1Δ-2 pep4 prb1 prc1) and CB018 (SOI1 pep4 prb1 prc1) carried pCWKX10-I718tail. Strains were labeled for 10 min (B and C) or 5 min (D), chased, and samples were collected at the indicated times after the addition of chase. Cells were lysed, and Kex2p was immunoprecipitated using antiserum raised against the Kex2p lumenal domain (55). WT Kex2p is indicated by closed arrows. I718tail Kex2p (B and C) and C-tailΔ Kex2p (D) are indicated by open arrows.

Mentions: Mutations in SOI1 were originally isolated as suppressors of the rapid loss of mating competence exhibited by a MATα strain after shutting off expression of Y713A Kex2p (34). Deletion of SOI1 also resulted in suppression (Fig. 7 A). All three original soi1 alleles also decreased the rate of delivery of Y713A Kex2p to the vacuole (34). In contrast, deletion of SOI1 did not effect on the rate of vacuolar delivery of Y713A Kex2p. The rate of vacuolar degradation of Y713A Kex2p was the same in SOI1 and soi1Δ strains (Fig. 6 A; t1/2 = 21 min ± 2 min [SEM] in soi1Δ strain, n = 4; t1/2 = 23 min ± 3 min in SOI1 strain, n = 4). The onset of impotence assay reflects the level of Kex2p activity in the pro-α-factor processing compartment, presumably the TGN, whereas the t1/2 of Kex2p may also reflect downstream events, e.g., the rate of Kex2p delivery from the PVC to the vacuole. Therefore, complete loss of Soi1p function apparently increased the concentration of Y713A Kex2p in the pro-α-factor processing compartment without measurably affecting the net rate of delivery of the protein to the vacuole. These results are consistent with a model in which Soi1p functions in two distinct steps in a pathway or cycle involved in Kex2p localization, one step governing the rate of transport of Kex2p from the PVC to the vacuole and the other affecting the concentration of the protein in the TGN.


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)

Deletion of SOI1 reveals a second TLS in the C-tail of  Kex2p. (A) BFY106-4D (SOI1 kex2Δ) and JBY134.1 (soi1Δ-1  kex2Δ) expressing WT Kex2p, Y713A Kex2p or I718tail Kex2p,  under the control of the GAL1 promoter, were shifted from galactose to glucose for the indicated times before testing mating  competence. (B and D) JBY135-1A (soi1Δ-1) and JBY135-2D  (SOI1) carried CEN plasmids expressing either I718tail Kex2p  (pCWKX10-I718tail; B) or C-tailΔ Kex2p (pCWKX17; D) from  the KEX2 promoter (34, 55). (C) JBY173 (soi1Δ-2 pep4 prb1  prc1) and CB018 (SOI1 pep4 prb1 prc1) carried pCWKX10-I718tail. Strains were labeled for 10 min (B and C) or 5 min (D),  chased, and samples were collected at the indicated times after  the addition of chase. Cells were lysed, and Kex2p was immunoprecipitated using antiserum raised against the Kex2p lumenal  domain (55). WT Kex2p is indicated by closed arrows. I718tail  Kex2p (B and C) and C-tailΔ Kex2p (D) are indicated by open  arrows.
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Figure 7: Deletion of SOI1 reveals a second TLS in the C-tail of Kex2p. (A) BFY106-4D (SOI1 kex2Δ) and JBY134.1 (soi1Δ-1 kex2Δ) expressing WT Kex2p, Y713A Kex2p or I718tail Kex2p, under the control of the GAL1 promoter, were shifted from galactose to glucose for the indicated times before testing mating competence. (B and D) JBY135-1A (soi1Δ-1) and JBY135-2D (SOI1) carried CEN plasmids expressing either I718tail Kex2p (pCWKX10-I718tail; B) or C-tailΔ Kex2p (pCWKX17; D) from the KEX2 promoter (34, 55). (C) JBY173 (soi1Δ-2 pep4 prb1 prc1) and CB018 (SOI1 pep4 prb1 prc1) carried pCWKX10-I718tail. Strains were labeled for 10 min (B and C) or 5 min (D), chased, and samples were collected at the indicated times after the addition of chase. Cells were lysed, and Kex2p was immunoprecipitated using antiserum raised against the Kex2p lumenal domain (55). WT Kex2p is indicated by closed arrows. I718tail Kex2p (B and C) and C-tailΔ Kex2p (D) are indicated by open arrows.
Mentions: Mutations in SOI1 were originally isolated as suppressors of the rapid loss of mating competence exhibited by a MATα strain after shutting off expression of Y713A Kex2p (34). Deletion of SOI1 also resulted in suppression (Fig. 7 A). All three original soi1 alleles also decreased the rate of delivery of Y713A Kex2p to the vacuole (34). In contrast, deletion of SOI1 did not effect on the rate of vacuolar delivery of Y713A Kex2p. The rate of vacuolar degradation of Y713A Kex2p was the same in SOI1 and soi1Δ strains (Fig. 6 A; t1/2 = 21 min ± 2 min [SEM] in soi1Δ strain, n = 4; t1/2 = 23 min ± 3 min in SOI1 strain, n = 4). The onset of impotence assay reflects the level of Kex2p activity in the pro-α-factor processing compartment, presumably the TGN, whereas the t1/2 of Kex2p may also reflect downstream events, e.g., the rate of Kex2p delivery from the PVC to the vacuole. Therefore, complete loss of Soi1p function apparently increased the concentration of Y713A Kex2p in the pro-α-factor processing compartment without measurably affecting the net rate of delivery of the protein to the vacuole. These results are consistent with a model in which Soi1p functions in two distinct steps in a pathway or cycle involved in Kex2p localization, one step governing the rate of transport of Kex2p from the PVC to the vacuole and the other affecting the concentration of the protein in the TGN.

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