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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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SOI1 is required for TGN localization of Kex2p,  Vps10p, and Ste13p, and for vacuolar targeting of proCPY. (A)  Pulse-chase/immunoprecipitation of Kex2p. BFY106-4D (kex2Δ  SOI1) and JBY134.1 (kex2Δ soi1Δ-1), transformed with CEN  plasmids expressing either WT Kex2p (pCWKX10) or Y713A  Kex2p (pCWKX11) from the KEX2 promoter (55), were pulse  labeled with [35S]H2SO4 for 10 min before addition of chase. Cells  were collected at the indicated times after addition of chase, lysed,  and processed for immunoprecipitation of Kex2p. (B) BFY106-4D and JBY134.1 were labeled as described in A and chased.  Samples were collected at the indicated times, separated into  cells (I, intracellular) and medium (E, extracellular), and processed for immunoprecipitation of CPY. Forms of CPY are as  follows: p1CPY, core-glycosylated ER form; p2CPY, Golgi form;  mCPY, mature vacuolar form (47). (C and D) JBY135-1A  (soi1Δ-1) and JBY135-2D (SOI1) were labeled with [35S]H2SO4  for 10 min and then chased. Samples were collected, lysed, and  immunoprecipitated using anti-Vps10p (C) or anti-ALP (D) sera.  Vps10p (C) and precursor A-ALP (D) were quantified after  SDS-PAGE and plotted relative to initial band intensity. Half-life (C) or half-time of processing (D) were determined by linear  regression (Table II).
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Figure 6: SOI1 is required for TGN localization of Kex2p, Vps10p, and Ste13p, and for vacuolar targeting of proCPY. (A) Pulse-chase/immunoprecipitation of Kex2p. BFY106-4D (kex2Δ SOI1) and JBY134.1 (kex2Δ soi1Δ-1), transformed with CEN plasmids expressing either WT Kex2p (pCWKX10) or Y713A Kex2p (pCWKX11) from the KEX2 promoter (55), were pulse labeled with [35S]H2SO4 for 10 min before addition of chase. Cells were collected at the indicated times after addition of chase, lysed, and processed for immunoprecipitation of Kex2p. (B) BFY106-4D and JBY134.1 were labeled as described in A and chased. Samples were collected at the indicated times, separated into cells (I, intracellular) and medium (E, extracellular), and processed for immunoprecipitation of CPY. Forms of CPY are as follows: p1CPY, core-glycosylated ER form; p2CPY, Golgi form; mCPY, mature vacuolar form (47). (C and D) JBY135-1A (soi1Δ-1) and JBY135-2D (SOI1) were labeled with [35S]H2SO4 for 10 min and then chased. Samples were collected, lysed, and immunoprecipitated using anti-Vps10p (C) or anti-ALP (D) sera. Vps10p (C) and precursor A-ALP (D) were quantified after SDS-PAGE and plotted relative to initial band intensity. Half-life (C) or half-time of processing (D) were determined by linear regression (Table II).

Mentions: The original soi1 mutant alleles accelerated the degradation of WT Kex2p (34). To examine the effect of the soi1Δ mutation on the vacuolar degradation of WT Kex2p, we measured the t1/2 of the protein in the soi1Δ strain. As in the case of the original soi1 alleles, WT Kex2p was degraded more rapidly in the soi1Δ strain (Fig. 6 A; t1/2 = 50 min ± 4 min [SEM], n = 3) than in the wild-type strain (t1/2 = 112 min ± 10 min, n = 3). Therefore, localization of WT Kex2p was perturbed by the soi1Δ mutation in the same way as by the original mutant alleles.


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)

SOI1 is required for TGN localization of Kex2p,  Vps10p, and Ste13p, and for vacuolar targeting of proCPY. (A)  Pulse-chase/immunoprecipitation of Kex2p. BFY106-4D (kex2Δ  SOI1) and JBY134.1 (kex2Δ soi1Δ-1), transformed with CEN  plasmids expressing either WT Kex2p (pCWKX10) or Y713A  Kex2p (pCWKX11) from the KEX2 promoter (55), were pulse  labeled with [35S]H2SO4 for 10 min before addition of chase. Cells  were collected at the indicated times after addition of chase, lysed,  and processed for immunoprecipitation of Kex2p. (B) BFY106-4D and JBY134.1 were labeled as described in A and chased.  Samples were collected at the indicated times, separated into  cells (I, intracellular) and medium (E, extracellular), and processed for immunoprecipitation of CPY. Forms of CPY are as  follows: p1CPY, core-glycosylated ER form; p2CPY, Golgi form;  mCPY, mature vacuolar form (47). (C and D) JBY135-1A  (soi1Δ-1) and JBY135-2D (SOI1) were labeled with [35S]H2SO4  for 10 min and then chased. Samples were collected, lysed, and  immunoprecipitated using anti-Vps10p (C) or anti-ALP (D) sera.  Vps10p (C) and precursor A-ALP (D) were quantified after  SDS-PAGE and plotted relative to initial band intensity. Half-life (C) or half-time of processing (D) were determined by linear  regression (Table II).
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Figure 6: SOI1 is required for TGN localization of Kex2p, Vps10p, and Ste13p, and for vacuolar targeting of proCPY. (A) Pulse-chase/immunoprecipitation of Kex2p. BFY106-4D (kex2Δ SOI1) and JBY134.1 (kex2Δ soi1Δ-1), transformed with CEN plasmids expressing either WT Kex2p (pCWKX10) or Y713A Kex2p (pCWKX11) from the KEX2 promoter (55), were pulse labeled with [35S]H2SO4 for 10 min before addition of chase. Cells were collected at the indicated times after addition of chase, lysed, and processed for immunoprecipitation of Kex2p. (B) BFY106-4D and JBY134.1 were labeled as described in A and chased. Samples were collected at the indicated times, separated into cells (I, intracellular) and medium (E, extracellular), and processed for immunoprecipitation of CPY. Forms of CPY are as follows: p1CPY, core-glycosylated ER form; p2CPY, Golgi form; mCPY, mature vacuolar form (47). (C and D) JBY135-1A (soi1Δ-1) and JBY135-2D (SOI1) were labeled with [35S]H2SO4 for 10 min and then chased. Samples were collected, lysed, and immunoprecipitated using anti-Vps10p (C) or anti-ALP (D) sera. Vps10p (C) and precursor A-ALP (D) were quantified after SDS-PAGE and plotted relative to initial band intensity. Half-life (C) or half-time of processing (D) were determined by linear regression (Table II).
Mentions: The original soi1 mutant alleles accelerated the degradation of WT Kex2p (34). To examine the effect of the soi1Δ mutation on the vacuolar degradation of WT Kex2p, we measured the t1/2 of the protein in the soi1Δ strain. As in the case of the original soi1 alleles, WT Kex2p was degraded more rapidly in the soi1Δ strain (Fig. 6 A; t1/2 = 50 min ± 4 min [SEM], n = 3) than in the wild-type strain (t1/2 = 112 min ± 10 min, n = 3). Therefore, localization of WT Kex2p was perturbed by the soi1Δ mutation in the same way as by the original mutant alleles.

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