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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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Mapping TLS2. A  series of Kex2p truncation  mutants (shown schematically in Fig. 1) was expressed  under the control of the  GAL1 promoter in JBY154-1A (MATα kex2Δ soi1Δ-2)  and JBY154-2A (MATα  kex2Δ SOI1), and was tested  for mating competence in the  onset of impotence assay after 10 h on glucose. Sequence  analysis of the 778tail Kex2p  used in this experiment revealed that this protein had a  second mutation at the carboxy terminus (P778S). This  second mutation was inconsequential in that other isolates of this truncation that  lacked this second mutation  behaved identically to the  one shown (data not shown).
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Figure 11: Mapping TLS2. A series of Kex2p truncation mutants (shown schematically in Fig. 1) was expressed under the control of the GAL1 promoter in JBY154-1A (MATα kex2Δ soi1Δ-2) and JBY154-2A (MATα kex2Δ SOI1), and was tested for mating competence in the onset of impotence assay after 10 h on glucose. Sequence analysis of the 778tail Kex2p used in this experiment revealed that this protein had a second mutation at the carboxy terminus (P778S). This second mutation was inconsequential in that other isolates of this truncation that lacked this second mutation behaved identically to the one shown (data not shown).

Mentions: To map the Kex2p TLS2, we examined a series of truncated forms of Kex2p in the soi1Δ strain using the onset of impotence assay. These truncation mutants were indistinguishable from full-length Kex2p in the SOI1 strain (Fig. 11). In the soi1Δ strain, however, mating behavior indicated that whereas 778tail Kex2p possessed TLS2, its function was lost by deletion of six additional residues (see 772tail Kex2p in Fig. 11). Thus, the COOH-terminal endpoint of TLS2 appears to be discrete.


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)

Mapping TLS2. A  series of Kex2p truncation  mutants (shown schematically in Fig. 1) was expressed  under the control of the  GAL1 promoter in JBY154-1A (MATα kex2Δ soi1Δ-2)  and JBY154-2A (MATα  kex2Δ SOI1), and was tested  for mating competence in the  onset of impotence assay after 10 h on glucose. Sequence  analysis of the 778tail Kex2p  used in this experiment revealed that this protein had a  second mutation at the carboxy terminus (P778S). This  second mutation was inconsequential in that other isolates of this truncation that  lacked this second mutation  behaved identically to the  one shown (data not shown).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 11: Mapping TLS2. A series of Kex2p truncation mutants (shown schematically in Fig. 1) was expressed under the control of the GAL1 promoter in JBY154-1A (MATα kex2Δ soi1Δ-2) and JBY154-2A (MATα kex2Δ SOI1), and was tested for mating competence in the onset of impotence assay after 10 h on glucose. Sequence analysis of the 778tail Kex2p used in this experiment revealed that this protein had a second mutation at the carboxy terminus (P778S). This second mutation was inconsequential in that other isolates of this truncation that lacked this second mutation behaved identically to the one shown (data not shown).
Mentions: To map the Kex2p TLS2, we examined a series of truncated forms of Kex2p in the soi1Δ strain using the onset of impotence assay. These truncation mutants were indistinguishable from full-length Kex2p in the SOI1 strain (Fig. 11). In the soi1Δ strain, however, mating behavior indicated that whereas 778tail Kex2p possessed TLS2, its function was lost by deletion of six additional residues (see 772tail Kex2p in Fig. 11). Thus, the COOH-terminal endpoint of TLS2 appears to be discrete.

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