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Proteolytic processing of a precursor protein for a growth-promoting peptide by a subtilisin serine protease in Arabidopsis.

Srivastava R, Liu JX, Howell SH - Plant J. (2008)

Bottom Line: The processing of proAtPSK4 was dependent on AtSBT1.1, a subtilisin-like serine protease, encoded by one of 56 subtilase genes in Arabidopsis.We also demonstrated that a fluorogenic peptide representing the putative subtilase recognition site in proAtPSK4 is cleaved in vitro by affinity-purified AtSBT1.1.An alanine scan through the recognition site peptide indicated that AtSBT1.1 is fairly specific for the AtPSK4 precursor.

View Article: PubMed Central - PubMed

Affiliation: Plant Sciences Institute, Iowa State University, Ames, IA 50011, USA.

ABSTRACT
Phytosulfokines (PSKs) are secreted, sulfated peptide hormones derived from larger prepropeptide precursors. Proteolytic processing of one of the precursors, AtPSK4, was demonstrated by cleavage of a preproAtPSK4-myc transgene product to AtPSK4-myc. Cleavage of proAtPSK4 was induced by placing root explants in tissue culture. The processing of proAtPSK4 was dependent on AtSBT1.1, a subtilisin-like serine protease, encoded by one of 56 subtilase genes in Arabidopsis. The gene encoding AtSBT1.1 was up-regulated following the transfer of root explants to tissue culture, suggesting that activation of the proteolytic machinery that cleaves proAtPSK4 is dependent on AtSBT1.1 expression. We also demonstrated that a fluorogenic peptide representing the putative subtilase recognition site in proAtPSK4 is cleaved in vitro by affinity-purified AtSBT1.1. An alanine scan through the recognition site peptide indicated that AtSBT1.1 is fairly specific for the AtPSK4 precursor. Thus, this peptide growth factor, which promotes callus formation in culture, is proteolytically cleaved from its precursor by a specific plant subtilase encoded by a gene that is up-regulated during the process of transferring root explants to tissue culture.

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In vivo cleavage of pAtPSK4-myc.(a) Root segments from wild-type transgenic seedlings expressing the construct 35S:ppAtPSK4-myc were explanted and incubated on callus induction medium (CIM) for 1-4 days and then transferred onto shoot induction medium (SIM). Root segments from sbt1.1-1 and sbt1.1-2 mutants expressing 35S:ppAtPSK4-myc were also explanted and similarly incubated. Arrows indicate the predicted migration position for processed AtPSK4–myc. The lane marked NT is an extract from roots of non-transformed seedlings.(b) Root segments from seedlings bearing 35S:ppAtPSK4-myc in a wild-type background were explanted and incubated on normal CIM or on B5 basal medium, without cytokinin or auxin hormones.(c) Time course following explantation for acquisition of capacity to process pAtPSK4–myc on CIM medium.
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fig01: In vivo cleavage of pAtPSK4-myc.(a) Root segments from wild-type transgenic seedlings expressing the construct 35S:ppAtPSK4-myc were explanted and incubated on callus induction medium (CIM) for 1-4 days and then transferred onto shoot induction medium (SIM). Root segments from sbt1.1-1 and sbt1.1-2 mutants expressing 35S:ppAtPSK4-myc were also explanted and similarly incubated. Arrows indicate the predicted migration position for processed AtPSK4–myc. The lane marked NT is an extract from roots of non-transformed seedlings.(b) Root segments from seedlings bearing 35S:ppAtPSK4-myc in a wild-type background were explanted and incubated on normal CIM or on B5 basal medium, without cytokinin or auxin hormones.(c) Time course following explantation for acquisition of capacity to process pAtPSK4–myc on CIM medium.

Mentions: To determine whether AtSBT1.1 is involved in the proteolytic processing of AtPSKs, we developed a constitutively expressed, C-terminal 4 x myc-tagged construct of the AtPSK4 precursor, 35S:ppAtPSK4–myc, and studied its processing in vivo. PreproAtPSK4 will be referred to as ppAtPSK4 and proAtPSK4 as pAtPSK4. AtPSK4 was chosen for study because it is the most abundantly expressed PSK precursor (Matsubayashi and Sakagami, 2006). The predicted size of pAtPSK4 with the myc tag is 12.8 kDa, but we observed a band at approximately 19 kDa on Western blots, larger than the predicted size (Figure 1a, wt, 0 time). To demonstrate, nonetheless, that this band is pAtPSK4–myc, we analyzed the partially purified myc-tagged protein and identified three peptides derived from the fusion protein by MS/MS analysis (Figure S1). Thus, the larger apparent size of pAtPSK4–myc may be due to anomalous gel migration behavior or post-translational modification of part of the precursor protein.


Proteolytic processing of a precursor protein for a growth-promoting peptide by a subtilisin serine protease in Arabidopsis.

Srivastava R, Liu JX, Howell SH - Plant J. (2008)

In vivo cleavage of pAtPSK4-myc.(a) Root segments from wild-type transgenic seedlings expressing the construct 35S:ppAtPSK4-myc were explanted and incubated on callus induction medium (CIM) for 1-4 days and then transferred onto shoot induction medium (SIM). Root segments from sbt1.1-1 and sbt1.1-2 mutants expressing 35S:ppAtPSK4-myc were also explanted and similarly incubated. Arrows indicate the predicted migration position for processed AtPSK4–myc. The lane marked NT is an extract from roots of non-transformed seedlings.(b) Root segments from seedlings bearing 35S:ppAtPSK4-myc in a wild-type background were explanted and incubated on normal CIM or on B5 basal medium, without cytokinin or auxin hormones.(c) Time course following explantation for acquisition of capacity to process pAtPSK4–myc on CIM medium.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2667306&req=5

fig01: In vivo cleavage of pAtPSK4-myc.(a) Root segments from wild-type transgenic seedlings expressing the construct 35S:ppAtPSK4-myc were explanted and incubated on callus induction medium (CIM) for 1-4 days and then transferred onto shoot induction medium (SIM). Root segments from sbt1.1-1 and sbt1.1-2 mutants expressing 35S:ppAtPSK4-myc were also explanted and similarly incubated. Arrows indicate the predicted migration position for processed AtPSK4–myc. The lane marked NT is an extract from roots of non-transformed seedlings.(b) Root segments from seedlings bearing 35S:ppAtPSK4-myc in a wild-type background were explanted and incubated on normal CIM or on B5 basal medium, without cytokinin or auxin hormones.(c) Time course following explantation for acquisition of capacity to process pAtPSK4–myc on CIM medium.
Mentions: To determine whether AtSBT1.1 is involved in the proteolytic processing of AtPSKs, we developed a constitutively expressed, C-terminal 4 x myc-tagged construct of the AtPSK4 precursor, 35S:ppAtPSK4–myc, and studied its processing in vivo. PreproAtPSK4 will be referred to as ppAtPSK4 and proAtPSK4 as pAtPSK4. AtPSK4 was chosen for study because it is the most abundantly expressed PSK precursor (Matsubayashi and Sakagami, 2006). The predicted size of pAtPSK4 with the myc tag is 12.8 kDa, but we observed a band at approximately 19 kDa on Western blots, larger than the predicted size (Figure 1a, wt, 0 time). To demonstrate, nonetheless, that this band is pAtPSK4–myc, we analyzed the partially purified myc-tagged protein and identified three peptides derived from the fusion protein by MS/MS analysis (Figure S1). Thus, the larger apparent size of pAtPSK4–myc may be due to anomalous gel migration behavior or post-translational modification of part of the precursor protein.

Bottom Line: The processing of proAtPSK4 was dependent on AtSBT1.1, a subtilisin-like serine protease, encoded by one of 56 subtilase genes in Arabidopsis.We also demonstrated that a fluorogenic peptide representing the putative subtilase recognition site in proAtPSK4 is cleaved in vitro by affinity-purified AtSBT1.1.An alanine scan through the recognition site peptide indicated that AtSBT1.1 is fairly specific for the AtPSK4 precursor.

View Article: PubMed Central - PubMed

Affiliation: Plant Sciences Institute, Iowa State University, Ames, IA 50011, USA.

ABSTRACT
Phytosulfokines (PSKs) are secreted, sulfated peptide hormones derived from larger prepropeptide precursors. Proteolytic processing of one of the precursors, AtPSK4, was demonstrated by cleavage of a preproAtPSK4-myc transgene product to AtPSK4-myc. Cleavage of proAtPSK4 was induced by placing root explants in tissue culture. The processing of proAtPSK4 was dependent on AtSBT1.1, a subtilisin-like serine protease, encoded by one of 56 subtilase genes in Arabidopsis. The gene encoding AtSBT1.1 was up-regulated following the transfer of root explants to tissue culture, suggesting that activation of the proteolytic machinery that cleaves proAtPSK4 is dependent on AtSBT1.1 expression. We also demonstrated that a fluorogenic peptide representing the putative subtilase recognition site in proAtPSK4 is cleaved in vitro by affinity-purified AtSBT1.1. An alanine scan through the recognition site peptide indicated that AtSBT1.1 is fairly specific for the AtPSK4 precursor. Thus, this peptide growth factor, which promotes callus formation in culture, is proteolytically cleaved from its precursor by a specific plant subtilase encoded by a gene that is up-regulated during the process of transferring root explants to tissue culture.

Show MeSH
Related in: MedlinePlus