- Email: [email protected]
Ddi1 and Related Proteins
Clan AA (A28) | 61. Ddi1 and Related Proteins
[9] Richins, R.D. (1993). Organization and expression of the peanut chlorotic streak virus genome. Thesis, University of Kentucky. [10] Richins, R.D., Scholthof, H.B., Shepherd, R.J. (1987). Sequence of figwort mosaic virus DNA (caulimovirus group). Nucleic Acids Res. 15, 8451 8466. [11] Calvert, L.A., Ospina, M.D., Shepherd, R.J. (1995). Characterization of cassava vein mosaic virus: a distinct plant pararetrovirus. J. Gen. Virol. 76, 1271 1276. [12] Medberry, S.L., Lockhart, B.E.L., Olszewski, N.E. (1990). Properties of Commelina yellow mottle virus’s complete DNA sequence, genomic discontinuities and transcript suggest that it is a pararetrovirus. Nucleic Acids Res. 18, 5505 5512. [13] Hay, J.M., Jones, M.C., Blackebrough, M.L., Dasgupta, I., Davies, J.W., Hull, R. (1991). An analysis of the sequence of an infectious clone of rice tungro bacilliform virus, a plant pararetrovirus. Nucleic Acids Res. 19, 2615 2621.
255
[14] Qu, R., Bhattacharyya, M., Laco, G.S., de Kochko, A., Subba Rao, B.L., Kaniewska, M.B., Elmer, J.S., Rochester, D.E., Smith, C.E., Beachy, R.N. (1991). Characterization of the genome of rice tungro bacilliform virus: comparison with commelina yellow mottle virus and caulimoviruses. Virology 185, 354 364. [15] Laco, G.S., Kent, S.B.H., Beachy, R.N. (1995). Analysis of the proteolytic processing and activation of the rice tungro bacilliform virus reverse transcription. Virology 208, 207 214. [16] Hagen, L.S., Jaquemont, M., Lepingle, A., Lot, H., Tepfer, M. (1993). Nucleotide sequence and genomic organization of cacao swollen shoot virus. Virology 196, 619 628. [17] Bouhida, M., Lockhart, B.E.L., Olszewski, N.E. (1993). An analysis of the complete sequence of sugarcane bacilliform virus genome infectious to banana and rice. J. Gen. Virol. 74, 15 22.
Thomas Hohn Friedrich Miescher Institute, PO Box 3543, CH-4002 Basel, Switzerland. Email: [email protected] This article is reproduced from the previous edition, Volume 1, pp. 195 197, r 2004, Elsevier Ltd., with revisions made by the Editors. © 2013 Elsevier Ltd. All rights reserved. DOI: http://dx.doi.org/10.1016/B978-0-12-382219-2.00060-0
Handbook of Proteolytic Enzymes, 3rd Edn ISBN: 978-0-12-382219-2
Chapter 61
Ddi1 and Related Proteins DATABANKS MEROPS name: DNA-damage inducible protein 1 MEROPS classification: clan AA, family A28, peptidase A28.001 Species distribution: superkingdom Eukaryota Reference sequence from: Saccharomyces cerevisiae (UniProt: P40087)
described from other species. The existence of genes encoding related proteins (described as Ddi1p orthologs in all eukaryotic genomes [3,4]) established this as an important family of proteins with a retroviral proteinase (RVP), aspartic proteinase-like domain containing the typical active site motif Asp-Thr/Ser-Gly.
Activity and Specificity Name and History The Saccharomyces cerevisiae protein product of the YER143w gene has been designated (v-SNARE-master 1) (Vsm1) [1] for its role in SNARE interactions and protein secretion but has also been named (DNA damage inducible) (Ddi1) since expression is upregulated in response to DNA damage [2]. The latter name appears to have now been generally adopted for the S. cerevisiae protein and, by extension, ‘Ddi1-like’ proteins have been
The proteolytic activity of Ddi1 and related proteins is, as yet, unproven but it has clear structural features of the aspartic proteinase family and there is circumstantial evidence that its putative catalytic triad, and thus its proposed catalytic activity, is important in a number of its functions. No substrate or conditions for the enzymological assay of this protein have yet been established. Nonetheless, the production of active site mutants of the Ddi1 protein and analysis of their effects does provide some circumstantial evidence that catalytic activity is
256
important in its function in checkpoint regulation [5] and in regulating protein secretion [4]. Although no direct enzymological assay has been reported, the use of an indirect assay whereby Ddi1 knockout yeast were complemented by plasmid-borne genes encoding Ddi1-like proteins of S. cerevisiae, Leishmania major or human origin has been described [6]. This work indicated that the Leishmania variant may be the target of action of HIV proteinase inhibitors that have been demonstrated to affect growth, proliferation and survival of Leishmania parasites in culture and in patients receiving highly active retroviral therapy (HAART). A variety of HIV proteinase inhibitors were shown to have differential effects on each of the Ddi1like proteins, demonstrating the selectivity of each protein for a subset of the inhibitors and distinct activities for individual inhibitors against each family member.
Structural Chemistry Ddi1 from the yeast Saccharomyces cerevisiae is the best characterized member the family. In these proteins a highly conserved retroviral proteinase-like domain (RVPlike domain) is located within a longer protein sequence. In the case of some variants (e.g. from some Leishmania parasites) there is little flanking sequence whereas the RVP-like domain of the S. cerevisiae protein represents approximately the central third of the protein sequence. The S. cerevisiae protein is a member of the group of UBL-UBA proteins (Ddi1, Dsk2 and Rad23) and the sequence can be divided into several regions. At the Nterminus is a ubiquitin-like sequence (UBL, Bresidues 1 75); the second domain is the RVP-like domain, (Bresidues 191 324); this is followed by the C-terminal segment (Bresidues 325 428) containing a t-SNARE binding region (Bresidues 344 395) and a ubiquitin associated (UBA) domain at its C-terminus (Bresidues 392 428). The crystal structure of the RVP-like domain alone of ˚ resoluS. cerevisiae Ddi1 has been determined to 2.3 A tion (PDB entry 2I1A) [7]. A homodimeric structure is seen with a clear aspartic proteinase family fold. Homodimerization of Ddi1 has previously been shown to be independent of the presence of the UBA domain [8] and relies on the RVP region [5]. This is confirmed by the presence of the dimeric structure in solution and under crystallographic conditions [7]. The active site Asp-ThrGly motif of the Ddi1 RVP region has identical geometry to that of HIV proteinase. However, the active site trench in this structure is considerably wider than for other aspartic proteinases and, at present, it is unclear whether this is a feature of the protein or a crystallographic
Clan AA (A28) | 61. Ddi1 and Related Proteins
artifact. The β-hairpin ‘flap’ that overlies the active site in aspartic proteinases is not visible in the Ddi1 structure but the walls of the trench can be seen to be lined with hydrophobic residues, perhaps indicating that a hydrophobic substrate may be favored. Although the structure most closely resembles those of A2 family retroviral aspartic proteinases, the six-stranded β-sheet structure underlying the active site is made up of three strands from one monomer followed by three from the other. This is similar to the structure found in A1, pepsin family proteinases and is distinct from the typical retroviral pattern in which alternating strands from the two monomers interdigitate to form the six-strand sheet. The structure of the retroviral proteinase from xenotrophic murine leukemia virusrelated virus is atypical in this regard and shows similarity to the Ddi1 protein [9]. While most members of the Ddi1 family show the classical active site Asp-Thr-Gly or Asp-Ser-Gly motif, some variants, e.g. from Trypanosome parasites, contain an Asp-Cys-Gly sequence at this point [4]. Although the catalytic activity of these variants, as for other family members, is unproven, the Cys containing variants may represent the first identified naturally occurring forms of this motif, which has previously been shown to be catalytically viable in HIV proteinase mutants [10].
Preparation The RVP domain residues 180 325 from the S. cerevisiae Ddi1 protein have been expressed in E. coli as a soluble protein in this bacterium [7] and was used in the crystallographic studies described above. Soluble fractions from induced cell lysates were applied to SP Sepharose and eluted in a 50 to 500 mM NaCl gradient in 10 mM KPO4 buffer (pH 6.5) and fractions containing Ddi1 were dialyzed against 10 mM KPO4 buffer (pH 8.0) containing 50 mM NaCl.
Biological Aspects The DNA damage inducible protein Ddi1 of the yeast Saccharomyces cerevisiae is involved in a number of interactions with multiple proteins and has influence in protein targeting to the proteasome, control of cell cycle and suppression of protein secretion from the cell. The UBA domain appears to be important in the interactions of Ddi1 with some of its partner proteins such as Rad23 (a protein involved in nucleotide excision and repair) that forms a heterodimer with Ddi1 but not with a mutant Ddi1 lacking the UBA region [8]. Deletion of the UBA region also renders cells defective in S-phase
Clan AA (A28) | 61. Ddi1 and Related Proteins
checkpoint control [11]. The UBA domains of both Ddi1 and Rad23 interact directly with ubiquitin [12] and the binding of mono-ubiquitin requires the dissociation of the Ddi1-Rad23 heterodimers [13]. The Ddi1 UBA domain also interacts with ubiquitinated Ho protein and the UBL domain binds with the proteasome to deliver the Ho protein to this complex [14]. The UBL domains of both Ddi1 [14] and Rad23 [12] associate with the proteasome and have a role in the control of cell cycle by mediating the degradation of Pds1 by the proteasome to allow the onset of anaphase. UBL domains are present in the majority of Ddi1-like proteins but are absent from some family members [4]. There is some redundancy in the Ddi1/Rad23 regulation of cell cycle and dimerization of Ddi1 is necessary for its cell cycle functions [15]. Ddi1 also interacts with E3 ubiquitin-protein ligase SCF complexes that have a role in the regulation of G1/S cell cycle progression [16] and it appears that Ddi1 is involved in degradation of the F-box protein UFO. Ddi1 and Rad23 are both involved as negative regulators of the phosphateresponsive PHO pathway, interacting in their phosphorylated states with Pho81p [17]. In this case, however, Ddi1 and Rad23 do not appear to be involved in the degradation of Pho81p. The role of Ddi1 in protein secretion may be mediated via SNARE proteins making it a negative regulator of secretion. Ddi1 appears to interact with the Snc2 v-SNARE of the yeast late secretory pathway and Ddi1 knockout mutants lead to an increase in protein secretion to the medium [1]. The UBA domain amino acids 344 395 are important for interactions with Sso1 t-SNARE and this region is phosphorylated at T346 and T348 with the latter being necessary for Sso1 binding [5]. Phosphorylation of Sso exocytic t-SNAREs by protein kinase A also promotes binding of Ddi1, increasing its affinity more than five-fold. The binding of Ddi1 to Sso1 competes with the binding of the t-SNARE Sec9 so Ddi1 is proposed as an inhibitor of SNARE assembly in yeast [18]. However, deletion of the UBA domain from Ddi1 has no effect on protein secretion from yeast cells whereas both the N-terminal domain (containing the UBL sequence) and the putative active site aspartate residue have roles in regulation of secretion by the S. cerevisiae variant [4].
Related Peptidases As detailed above, Ddi1-family proteins are found in all eukaryotic genomes and show a high degree of sequence conservation in their RVP-like domains [3,4].
257
References [1] Lustgarten, V., Gerst, J.E. (1999). Yeast VSM1 encodes a v-SNARE binding protein that may act as a negative regulator of constitutive exocytosis. Mol. Cell. Biol. 19, 4480 4494. [2] Liu, Y., Xiao, W. (1997). Bidirectional regulation of two DNAdamage-inducible genes, MAG1 and DDI1, from Saccharomyces cerevisiae. Mol. Microbiol. 23, 777 789. [3] Krylov, D.M., Koonin, E.V. (2001). A novel family of predicted retroviral-like aspartyl proteases with a possible key role in eukaryotic cell cycle control. Curr. Biol. 11, R584 R587. [4] White, R.E., Dickinson, J.R., Semple, C.A.M., Powell, D.J., Berry, C. (2011). The retroviral proteinase active site and the Nterminus of Ddi1 are required for repression of protein secretion. FEBS Lett. 585, 139 142. [5] Gabriely, G., Kama, R., Gelin-Licht, R., Gerst, J.E. (2008). Different domains of the UBL-UBA ubiquitin receptor, Ddi1/ Vsm1, are involved in its multiple cellular roles. Mol. Biol. Cell. 19, 3625 3637. [6] White, R.E., Powell, D.J., Berry, C. (2011). HIV proteinase inhibitors target the Ddi1-like protein of Leishmania parasites. FASEB J. 25, 1729 1736. [7] Sirkis, R., Gerst, J.E., Fass, D. (2006). Ddi1, a eukaryotic protein with the retroviral protease fold. J. Mol. Biol. 364, 376 387. [8] Bertolaet, B.L., Clarke, D.J., Wolff, M., Watson, M.H., Henze, M., Divita, G., Reed, S.I. (2001). UBA domains mediate protein-protein interactions between two DNA damage-inducible proteins. J. Mol. Biol. 313, 955 963. [9] Li, M., Dimaio, F., Zhou, D., Gustchina, A., Lubkowski, J., Dauter, Z., Baker, D., Wlodawer, A. (2011). Crystal structure of XMRV protease differs from the structures of other retropepsins. Nat. Struct. Mol. Biol. 18(2), 227 279. [10] Strisovsky, K., Tessmer, U., Langner, J., Konvalinka, J., Krausslich, H.G. (2000). Systematic mutational analysis of the active-site threonine of HIV-1 proteinase, rethinking the ‘fireman’s grip’ hypothesis. Protein Sci. 9, 1631 1641. [11] Clarke, D.J., Mondesert, G., Segal, M., Bertolaet, B.L., Jensen, S., Wolff, M., Henze, M., Reed, S.I. (2001). Dosage suppressors of pds1 implicate ubiquitin-associated domains in checkpoint control. Mol. Cell. Biol. 21, 1997 2007. [12] Bertolaet, B.L., Clarke, D.J., Wolff, M., Watson, M.H., Henze, M., Divita, G., Reed, S.I. (2001). UBA domains of DNA damageinducible proteins interact with ubiquitin. Nat. Struct. Biol. 8, 417 422. [13] Kang, Y., Vossler, R.A., Diaz-Martinez, L.A., Winter, N.S., Clarke, D.J., Walters, K.J. (2006). UBL/UBA ubiquitin receptor proteins bind a common tetraubiquitin chain. J. Mol. Biol. 356, 1027 1035. [14] Kaplun, L., Tzirkin, R., Bakhrat, A., Shabek, N., Ivantsiv, Y., Raveh, D. (2005). The DNA damage-inducible UbL-UbA protein Ddi1 participates in Mec1-mediated degradation of Ho endonuclease. Mol. Cell. Biol. 25, 5355 5362. [15] Diaz-Martinez, L.A., Kang, Y., Walters, K.J., Clarke, D.J. (2006). Yeast UBL-UBA proteins have partially redundant functions in cell cycle control. Cell. Div. 1, 28. [16] Ivantsiv, Y., Kaplun, L., Tzirkin-Goldin, R., Shabek, N., Raveh, D. (2006). Unique role for the UbL-UbA protein Ddi1 in turnover of SCFUfo1 complexes. Mol. Cell. Biol. 26, 1579 1588.
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[17] Auesukaree, C., Fuchigami, I., Homma, T., Kaneko, Y., Harashima, S. (2008). Ddi1p and Rad23p play a cooperative role as negative regulators in the PHO pathway in Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 365, 821 825.
Clan AA (A28) | 61. Ddi1 and Related Proteins
[18] Marash, M., Gerst, J.E. (2003). Phosphorylation of the autoinhibitory domain of the Sso t-SNAREs promotes binding of the Vsm1 SNARE regulator in yeast. Mol. Biol. Cell. 14, 3114 3125.
Colin Berry Cardiff School of Biosciences, Cardiff University, Park Place, Cardiff CF10 3AT, UK. Email: [email protected] Handbook of Proteolytic Enzymes, 3rd Edn ISBN: 978-0-12-382219-2
© 2013 Elsevier Ltd. All rights reserved. DOI: http://dx.doi.org/10.1016/B978-0-12-382219-2.00061-2
[9] Richins, R.D. (1993). Organization and expression of the peanut chlorotic streak virus genome. Thesis, University of Kentucky. [10] Richins, R.D., Scholthof, H.B., Shepherd, R.J. (1987). Sequence of figwort mosaic virus DNA (caulimovirus group). Nucleic Acids Res. 15, 8451 8466. [11] Calvert, L.A., Ospina, M.D., Shepherd, R.J. (1995). Characterization of cassava vein mosaic virus: a distinct plant pararetrovirus. J. Gen. Virol. 76, 1271 1276. [12] Medberry, S.L., Lockhart, B.E.L., Olszewski, N.E. (1990). Properties of Commelina yellow mottle virus’s complete DNA sequence, genomic discontinuities and transcript suggest that it is a pararetrovirus. Nucleic Acids Res. 18, 5505 5512. [13] Hay, J.M., Jones, M.C., Blackebrough, M.L., Dasgupta, I., Davies, J.W., Hull, R. (1991). An analysis of the sequence of an infectious clone of rice tungro bacilliform virus, a plant pararetrovirus. Nucleic Acids Res. 19, 2615 2621.
255
[14] Qu, R., Bhattacharyya, M., Laco, G.S., de Kochko, A., Subba Rao, B.L., Kaniewska, M.B., Elmer, J.S., Rochester, D.E., Smith, C.E., Beachy, R.N. (1991). Characterization of the genome of rice tungro bacilliform virus: comparison with commelina yellow mottle virus and caulimoviruses. Virology 185, 354 364. [15] Laco, G.S., Kent, S.B.H., Beachy, R.N. (1995). Analysis of the proteolytic processing and activation of the rice tungro bacilliform virus reverse transcription. Virology 208, 207 214. [16] Hagen, L.S., Jaquemont, M., Lepingle, A., Lot, H., Tepfer, M. (1993). Nucleotide sequence and genomic organization of cacao swollen shoot virus. Virology 196, 619 628. [17] Bouhida, M., Lockhart, B.E.L., Olszewski, N.E. (1993). An analysis of the complete sequence of sugarcane bacilliform virus genome infectious to banana and rice. J. Gen. Virol. 74, 15 22.
Thomas Hohn Friedrich Miescher Institute, PO Box 3543, CH-4002 Basel, Switzerland. Email: [email protected] This article is reproduced from the previous edition, Volume 1, pp. 195 197, r 2004, Elsevier Ltd., with revisions made by the Editors. © 2013 Elsevier Ltd. All rights reserved. DOI: http://dx.doi.org/10.1016/B978-0-12-382219-2.00060-0
Handbook of Proteolytic Enzymes, 3rd Edn ISBN: 978-0-12-382219-2
Chapter 61
Ddi1 and Related Proteins DATABANKS MEROPS name: DNA-damage inducible protein 1 MEROPS classification: clan AA, family A28, peptidase A28.001 Species distribution: superkingdom Eukaryota Reference sequence from: Saccharomyces cerevisiae (UniProt: P40087)
described from other species. The existence of genes encoding related proteins (described as Ddi1p orthologs in all eukaryotic genomes [3,4]) established this as an important family of proteins with a retroviral proteinase (RVP), aspartic proteinase-like domain containing the typical active site motif Asp-Thr/Ser-Gly.
Activity and Specificity Name and History The Saccharomyces cerevisiae protein product of the YER143w gene has been designated (v-SNARE-master 1) (Vsm1) [1] for its role in SNARE interactions and protein secretion but has also been named (DNA damage inducible) (Ddi1) since expression is upregulated in response to DNA damage [2]. The latter name appears to have now been generally adopted for the S. cerevisiae protein and, by extension, ‘Ddi1-like’ proteins have been
The proteolytic activity of Ddi1 and related proteins is, as yet, unproven but it has clear structural features of the aspartic proteinase family and there is circumstantial evidence that its putative catalytic triad, and thus its proposed catalytic activity, is important in a number of its functions. No substrate or conditions for the enzymological assay of this protein have yet been established. Nonetheless, the production of active site mutants of the Ddi1 protein and analysis of their effects does provide some circumstantial evidence that catalytic activity is
256
important in its function in checkpoint regulation [5] and in regulating protein secretion [4]. Although no direct enzymological assay has been reported, the use of an indirect assay whereby Ddi1 knockout yeast were complemented by plasmid-borne genes encoding Ddi1-like proteins of S. cerevisiae, Leishmania major or human origin has been described [6]. This work indicated that the Leishmania variant may be the target of action of HIV proteinase inhibitors that have been demonstrated to affect growth, proliferation and survival of Leishmania parasites in culture and in patients receiving highly active retroviral therapy (HAART). A variety of HIV proteinase inhibitors were shown to have differential effects on each of the Ddi1like proteins, demonstrating the selectivity of each protein for a subset of the inhibitors and distinct activities for individual inhibitors against each family member.
Structural Chemistry Ddi1 from the yeast Saccharomyces cerevisiae is the best characterized member the family. In these proteins a highly conserved retroviral proteinase-like domain (RVPlike domain) is located within a longer protein sequence. In the case of some variants (e.g. from some Leishmania parasites) there is little flanking sequence whereas the RVP-like domain of the S. cerevisiae protein represents approximately the central third of the protein sequence. The S. cerevisiae protein is a member of the group of UBL-UBA proteins (Ddi1, Dsk2 and Rad23) and the sequence can be divided into several regions. At the Nterminus is a ubiquitin-like sequence (UBL, Bresidues 1 75); the second domain is the RVP-like domain, (Bresidues 191 324); this is followed by the C-terminal segment (Bresidues 325 428) containing a t-SNARE binding region (Bresidues 344 395) and a ubiquitin associated (UBA) domain at its C-terminus (Bresidues 392 428). The crystal structure of the RVP-like domain alone of ˚ resoluS. cerevisiae Ddi1 has been determined to 2.3 A tion (PDB entry 2I1A) [7]. A homodimeric structure is seen with a clear aspartic proteinase family fold. Homodimerization of Ddi1 has previously been shown to be independent of the presence of the UBA domain [8] and relies on the RVP region [5]. This is confirmed by the presence of the dimeric structure in solution and under crystallographic conditions [7]. The active site Asp-ThrGly motif of the Ddi1 RVP region has identical geometry to that of HIV proteinase. However, the active site trench in this structure is considerably wider than for other aspartic proteinases and, at present, it is unclear whether this is a feature of the protein or a crystallographic
Clan AA (A28) | 61. Ddi1 and Related Proteins
artifact. The β-hairpin ‘flap’ that overlies the active site in aspartic proteinases is not visible in the Ddi1 structure but the walls of the trench can be seen to be lined with hydrophobic residues, perhaps indicating that a hydrophobic substrate may be favored. Although the structure most closely resembles those of A2 family retroviral aspartic proteinases, the six-stranded β-sheet structure underlying the active site is made up of three strands from one monomer followed by three from the other. This is similar to the structure found in A1, pepsin family proteinases and is distinct from the typical retroviral pattern in which alternating strands from the two monomers interdigitate to form the six-strand sheet. The structure of the retroviral proteinase from xenotrophic murine leukemia virusrelated virus is atypical in this regard and shows similarity to the Ddi1 protein [9]. While most members of the Ddi1 family show the classical active site Asp-Thr-Gly or Asp-Ser-Gly motif, some variants, e.g. from Trypanosome parasites, contain an Asp-Cys-Gly sequence at this point [4]. Although the catalytic activity of these variants, as for other family members, is unproven, the Cys containing variants may represent the first identified naturally occurring forms of this motif, which has previously been shown to be catalytically viable in HIV proteinase mutants [10].
Preparation The RVP domain residues 180 325 from the S. cerevisiae Ddi1 protein have been expressed in E. coli as a soluble protein in this bacterium [7] and was used in the crystallographic studies described above. Soluble fractions from induced cell lysates were applied to SP Sepharose and eluted in a 50 to 500 mM NaCl gradient in 10 mM KPO4 buffer (pH 6.5) and fractions containing Ddi1 were dialyzed against 10 mM KPO4 buffer (pH 8.0) containing 50 mM NaCl.
Biological Aspects The DNA damage inducible protein Ddi1 of the yeast Saccharomyces cerevisiae is involved in a number of interactions with multiple proteins and has influence in protein targeting to the proteasome, control of cell cycle and suppression of protein secretion from the cell. The UBA domain appears to be important in the interactions of Ddi1 with some of its partner proteins such as Rad23 (a protein involved in nucleotide excision and repair) that forms a heterodimer with Ddi1 but not with a mutant Ddi1 lacking the UBA region [8]. Deletion of the UBA region also renders cells defective in S-phase
Clan AA (A28) | 61. Ddi1 and Related Proteins
checkpoint control [11]. The UBA domains of both Ddi1 and Rad23 interact directly with ubiquitin [12] and the binding of mono-ubiquitin requires the dissociation of the Ddi1-Rad23 heterodimers [13]. The Ddi1 UBA domain also interacts with ubiquitinated Ho protein and the UBL domain binds with the proteasome to deliver the Ho protein to this complex [14]. The UBL domains of both Ddi1 [14] and Rad23 [12] associate with the proteasome and have a role in the control of cell cycle by mediating the degradation of Pds1 by the proteasome to allow the onset of anaphase. UBL domains are present in the majority of Ddi1-like proteins but are absent from some family members [4]. There is some redundancy in the Ddi1/Rad23 regulation of cell cycle and dimerization of Ddi1 is necessary for its cell cycle functions [15]. Ddi1 also interacts with E3 ubiquitin-protein ligase SCF complexes that have a role in the regulation of G1/S cell cycle progression [16] and it appears that Ddi1 is involved in degradation of the F-box protein UFO. Ddi1 and Rad23 are both involved as negative regulators of the phosphateresponsive PHO pathway, interacting in their phosphorylated states with Pho81p [17]. In this case, however, Ddi1 and Rad23 do not appear to be involved in the degradation of Pho81p. The role of Ddi1 in protein secretion may be mediated via SNARE proteins making it a negative regulator of secretion. Ddi1 appears to interact with the Snc2 v-SNARE of the yeast late secretory pathway and Ddi1 knockout mutants lead to an increase in protein secretion to the medium [1]. The UBA domain amino acids 344 395 are important for interactions with Sso1 t-SNARE and this region is phosphorylated at T346 and T348 with the latter being necessary for Sso1 binding [5]. Phosphorylation of Sso exocytic t-SNAREs by protein kinase A also promotes binding of Ddi1, increasing its affinity more than five-fold. The binding of Ddi1 to Sso1 competes with the binding of the t-SNARE Sec9 so Ddi1 is proposed as an inhibitor of SNARE assembly in yeast [18]. However, deletion of the UBA domain from Ddi1 has no effect on protein secretion from yeast cells whereas both the N-terminal domain (containing the UBL sequence) and the putative active site aspartate residue have roles in regulation of secretion by the S. cerevisiae variant [4].
Related Peptidases As detailed above, Ddi1-family proteins are found in all eukaryotic genomes and show a high degree of sequence conservation in their RVP-like domains [3,4].
257
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[17] Auesukaree, C., Fuchigami, I., Homma, T., Kaneko, Y., Harashima, S. (2008). Ddi1p and Rad23p play a cooperative role as negative regulators in the PHO pathway in Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 365, 821 825.
Clan AA (A28) | 61. Ddi1 and Related Proteins
[18] Marash, M., Gerst, J.E. (2003). Phosphorylation of the autoinhibitory domain of the Sso t-SNAREs promotes binding of the Vsm1 SNARE regulator in yeast. Mol. Biol. Cell. 14, 3114 3125.
Colin Berry Cardiff School of Biosciences, Cardiff University, Park Place, Cardiff CF10 3AT, UK. Email: [email protected] Handbook of Proteolytic Enzymes, 3rd Edn ISBN: 978-0-12-382219-2
© 2013 Elsevier Ltd. All rights reserved. DOI: http://dx.doi.org/10.1016/B978-0-12-382219-2.00061-2