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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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(A) Suppression of the localization defect of Y713A  Kex2p in the soi1Δ strain requires TLS2. JBY154-1A (MATα  soi1Δ-2 kex2Δ) and JBY154-2A (MATα SOI1 kex2Δ) expressing either Y713A Kex2p (pCWKX21), Y713A I718tail Kex2p  (pCWKX21-I718), or C-tailΔ Kex2p (pCWKX27) under the control of the GAL1 promoter on CEN plasmids were shifted from  galactose to glucose for 5 h before testing mating competence.  (B) Optimal TLS1 function requires Soi1p, but TLS1 exhibits residual function in the absence of Soi1p. Strains JBY154-1A and  JBY154-2A expressing either I718tail Kex2p (pCWKX20-I718)  or Y713A I718tail Kex2p under the control of the GAL1 promoter  on CEN plasmids were shifted from galactose to glucose medium  for the indicated times before testing mating competence. Indicated to the left of each row of patches is the form of Kex2p expressed. The relevant SOI1 allele is indicated above the columns.
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Figure 8: (A) Suppression of the localization defect of Y713A Kex2p in the soi1Δ strain requires TLS2. JBY154-1A (MATα soi1Δ-2 kex2Δ) and JBY154-2A (MATα SOI1 kex2Δ) expressing either Y713A Kex2p (pCWKX21), Y713A I718tail Kex2p (pCWKX21-I718), or C-tailΔ Kex2p (pCWKX27) under the control of the GAL1 promoter on CEN plasmids were shifted from galactose to glucose for 5 h before testing mating competence. (B) Optimal TLS1 function requires Soi1p, but TLS1 exhibits residual function in the absence of Soi1p. Strains JBY154-1A and JBY154-2A expressing either I718tail Kex2p (pCWKX20-I718) or Y713A I718tail Kex2p under the control of the GAL1 promoter on CEN plasmids were shifted from galactose to glucose medium for the indicated times before testing mating competence. Indicated to the left of each row of patches is the form of Kex2p expressed. The relevant SOI1 allele is indicated above the columns.

Mentions: The suppression of the mating defect of Y713A Kex2p in soi1Δ strains could be explained in two ways. It might have resulted from loss of active discrimination against Ala at position 713, which might also explain the defect in the function of the WT TLS1. Alternatively, suppression might have resulted from the activation of TLS2 function by removal of Soi1p. The first model predicts that suppression of Tyr713Ala should be identical in the context of either the full-length tail or the I718tail, whereas the second model predicts that suppression should depend on TLS2. To distinguish between these models, we substituted Ala for Tyr713 in I718tail Kex2p and assessed the TGN localization of this protein in SOI1 and soi1Δ strains using the onset of impotence assay. Y713A I718tail Kex2p behaved identically in SOI1 and soi1Δ strains in the onset of impotence assay (Fig. 8, A and B). Suppression of Y713A was only observed when TLS2 was present, i.e., only in the context of the full-length tail. Therefore, suppression of Y713A Kex2p in soi1 mutants resulted from activation of TLS2, implying that TLS2 is normally antagonized by Soi1p. Y713A-I718tail Kex2p, lacking both TLS1 and TLS2, behaved like C-tailΔ Kex2p in this assay (Fig. 8 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)

(A) Suppression of the localization defect of Y713A  Kex2p in the soi1Δ strain requires TLS2. JBY154-1A (MATα  soi1Δ-2 kex2Δ) and JBY154-2A (MATα SOI1 kex2Δ) expressing either Y713A Kex2p (pCWKX21), Y713A I718tail Kex2p  (pCWKX21-I718), or C-tailΔ Kex2p (pCWKX27) under the control of the GAL1 promoter on CEN plasmids were shifted from  galactose to glucose for 5 h before testing mating competence.  (B) Optimal TLS1 function requires Soi1p, but TLS1 exhibits residual function in the absence of Soi1p. Strains JBY154-1A and  JBY154-2A expressing either I718tail Kex2p (pCWKX20-I718)  or Y713A I718tail Kex2p under the control of the GAL1 promoter  on CEN plasmids were shifted from galactose to glucose medium  for the indicated times before testing mating competence. Indicated to the left of each row of patches is the form of Kex2p expressed. The relevant SOI1 allele is indicated above the columns.
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Figure 8: (A) Suppression of the localization defect of Y713A Kex2p in the soi1Δ strain requires TLS2. JBY154-1A (MATα soi1Δ-2 kex2Δ) and JBY154-2A (MATα SOI1 kex2Δ) expressing either Y713A Kex2p (pCWKX21), Y713A I718tail Kex2p (pCWKX21-I718), or C-tailΔ Kex2p (pCWKX27) under the control of the GAL1 promoter on CEN plasmids were shifted from galactose to glucose for 5 h before testing mating competence. (B) Optimal TLS1 function requires Soi1p, but TLS1 exhibits residual function in the absence of Soi1p. Strains JBY154-1A and JBY154-2A expressing either I718tail Kex2p (pCWKX20-I718) or Y713A I718tail Kex2p under the control of the GAL1 promoter on CEN plasmids were shifted from galactose to glucose medium for the indicated times before testing mating competence. Indicated to the left of each row of patches is the form of Kex2p expressed. The relevant SOI1 allele is indicated above the columns.
Mentions: The suppression of the mating defect of Y713A Kex2p in soi1Δ strains could be explained in two ways. It might have resulted from loss of active discrimination against Ala at position 713, which might also explain the defect in the function of the WT TLS1. Alternatively, suppression might have resulted from the activation of TLS2 function by removal of Soi1p. The first model predicts that suppression of Tyr713Ala should be identical in the context of either the full-length tail or the I718tail, whereas the second model predicts that suppression should depend on TLS2. To distinguish between these models, we substituted Ala for Tyr713 in I718tail Kex2p and assessed the TGN localization of this protein in SOI1 and soi1Δ strains using the onset of impotence assay. Y713A I718tail Kex2p behaved identically in SOI1 and soi1Δ strains in the onset of impotence assay (Fig. 8, A and B). Suppression of Y713A was only observed when TLS2 was present, i.e., only in the context of the full-length tail. Therefore, suppression of Y713A Kex2p in soi1 mutants resulted from activation of TLS2, implying that TLS2 is normally antagonized by Soi1p. Y713A-I718tail Kex2p, lacking both TLS1 and TLS2, behaved like C-tailΔ Kex2p in this assay (Fig. 8 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