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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) Improved TGN localization of F87A A-ALP in a  soi1 mutant strain. Strains JBY135-1A (soi1Δ-1) and JBY135-2D  (SOI1), transformed with pSN98 (expressing F87A A-ALP; reference 29), were labeled for 10 min with [35S]H2SO4 and chased for  120 min. Samples were collected after 10, 20, 30, 60, and 120 min  of chase and processed for immunoprecipitation using anti-ALP  antiserum. Band intensity of pro-A-ALP was quantified after  SDS-PAGE (see Materials and Methods) and plotted relative to  initial band intensity. (B) Deletion of SOI1 does not affect transport through early secretory compartments. Strains JBY154-1D  (KEX2 soi1Δ-2) and JBY154-8B (KEX2 SOI1) were labeled with  EXPRE35S35S label for 2 min before addition of chase. Cells were  collected at the indicated times after addition of chase. Indicated  beside the top panel are precursor forms of Kex2p, I1 (pro-Kex2p  possessing core glycosyl modifications) and I2 (mature, core glycosylated Kex2p), and Golgi-modified, mature Kex2p (J; reference 54). Indicated beside the bottom panel are proALP and mature ALP (21).
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Figure 9: (A) Improved TGN localization of F87A A-ALP in a soi1 mutant strain. Strains JBY135-1A (soi1Δ-1) and JBY135-2D (SOI1), transformed with pSN98 (expressing F87A A-ALP; reference 29), were labeled for 10 min with [35S]H2SO4 and chased for 120 min. Samples were collected after 10, 20, 30, 60, and 120 min of chase and processed for immunoprecipitation using anti-ALP antiserum. Band intensity of pro-A-ALP was quantified after SDS-PAGE (see Materials and Methods) and plotted relative to initial band intensity. (B) Deletion of SOI1 does not affect transport through early secretory compartments. Strains JBY154-1D (KEX2 soi1Δ-2) and JBY154-8B (KEX2 SOI1) were labeled with EXPRE35S35S label for 2 min before addition of chase. Cells were collected at the indicated times after addition of chase. Indicated beside the top panel are precursor forms of Kex2p, I1 (pro-Kex2p possessing core glycosyl modifications) and I2 (mature, core glycosylated Kex2p), and Golgi-modified, mature Kex2p (J; reference 54). Indicated beside the bottom panel are proALP and mature ALP (21).

Mentions: Previously, we found that the original soi1 mutations suppressed the effects of the F87A substitution in the Ste13p C-tail, as measured by the t1/2 of maturation of the A-ALP fusion protein (34). To determine conclusively whether suppression in this case resulted from loss of Soi1p function, we examined the t1/2 for maturation of F87A A-ALP in the soi1Δ strain (Fig. 9 A). In the SOI1 strain, F87A A-ALP matured with a t1/2 of ∼60 min (Fig. 9; references 29, 36). In the soi1Δ strain, maturation of F87A A-ALP was delayed, occurring with a t1/2 of 125 min (Fig. 9 and Table II). Thus, deletion of SOI1 resulted in a slower rate of delivery of F87A A-ALP to the vacuole, suggesting that loss of Soi1p resulted in activation of a second TGN localization signal in the Ste13p C-tail.


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) Improved TGN localization of F87A A-ALP in a  soi1 mutant strain. Strains JBY135-1A (soi1Δ-1) and JBY135-2D  (SOI1), transformed with pSN98 (expressing F87A A-ALP; reference 29), were labeled for 10 min with [35S]H2SO4 and chased for  120 min. Samples were collected after 10, 20, 30, 60, and 120 min  of chase and processed for immunoprecipitation using anti-ALP  antiserum. Band intensity of pro-A-ALP was quantified after  SDS-PAGE (see Materials and Methods) and plotted relative to  initial band intensity. (B) Deletion of SOI1 does not affect transport through early secretory compartments. Strains JBY154-1D  (KEX2 soi1Δ-2) and JBY154-8B (KEX2 SOI1) were labeled with  EXPRE35S35S label for 2 min before addition of chase. Cells were  collected at the indicated times after addition of chase. Indicated  beside the top panel are precursor forms of Kex2p, I1 (pro-Kex2p  possessing core glycosyl modifications) and I2 (mature, core glycosylated Kex2p), and Golgi-modified, mature Kex2p (J; reference 54). Indicated beside the bottom panel are proALP and mature ALP (21).
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Figure 9: (A) Improved TGN localization of F87A A-ALP in a soi1 mutant strain. Strains JBY135-1A (soi1Δ-1) and JBY135-2D (SOI1), transformed with pSN98 (expressing F87A A-ALP; reference 29), were labeled for 10 min with [35S]H2SO4 and chased for 120 min. Samples were collected after 10, 20, 30, 60, and 120 min of chase and processed for immunoprecipitation using anti-ALP antiserum. Band intensity of pro-A-ALP was quantified after SDS-PAGE (see Materials and Methods) and plotted relative to initial band intensity. (B) Deletion of SOI1 does not affect transport through early secretory compartments. Strains JBY154-1D (KEX2 soi1Δ-2) and JBY154-8B (KEX2 SOI1) were labeled with EXPRE35S35S label for 2 min before addition of chase. Cells were collected at the indicated times after addition of chase. Indicated beside the top panel are precursor forms of Kex2p, I1 (pro-Kex2p possessing core glycosyl modifications) and I2 (mature, core glycosylated Kex2p), and Golgi-modified, mature Kex2p (J; reference 54). Indicated beside the bottom panel are proALP and mature ALP (21).
Mentions: Previously, we found that the original soi1 mutations suppressed the effects of the F87A substitution in the Ste13p C-tail, as measured by the t1/2 of maturation of the A-ALP fusion protein (34). To determine conclusively whether suppression in this case resulted from loss of Soi1p function, we examined the t1/2 for maturation of F87A A-ALP in the soi1Δ strain (Fig. 9 A). In the SOI1 strain, F87A A-ALP matured with a t1/2 of ∼60 min (Fig. 9; references 29, 36). In the soi1Δ strain, maturation of F87A A-ALP was delayed, occurring with a t1/2 of 125 min (Fig. 9 and Table II). Thus, deletion of SOI1 resulted in a slower rate of delivery of F87A A-ALP to the vacuole, suggesting that loss of Soi1p resulted in activation of a second TGN localization signal in the Ste13p C-tail.

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