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Altered intracellular localization and mobility of SBDS protein upon mutation in Shwachman-Diamond syndrome.

Orelio C, van der Sluis RM, Verkuijlen P, Nethe M, Hordijk PL, van den Berg TK, Kuijpers TW - PLoS ONE (2011)

Bottom Line: Further studies with a series of SBDS mutant proteins revealed that three distinct motifs determine the intracellular mobility of SBDS protein.A sumoylation motif in the C-terminal domain, that is lacking in patient SBDS proteins, was found to play a pivotal role in intracellular motility.Our structure-function analyses provide new insight into localization and motility of the SBDS protein, and show that patient-related mutant proteins are altered in their molecular properties, which may contribute to the clinical features observed in SDS patients.

View Article: PubMed Central - PubMed

Affiliation: Sanquin Research and Landsteiner Laboratory of the Academic Medical Center, Department of Blood Cell Research, University of Amsterdam, Amsterdam, The Netherlands.

ABSTRACT
Shwachman-Diamond Syndrome (SDS) is a rare inherited disease caused by mutations in the SBDS gene. Hematopoietic defects, exocrine pancreas dysfunction and short stature are the most prominent clinical features. To gain understanding of the molecular properties of the ubiquitously expressed SBDS protein, we examined its intracellular localization and mobility by live cell imaging techniques. We observed that SBDS full-length protein was localized in both the nucleus and cytoplasm, whereas patient-related truncated SBDS protein isoforms localize predominantly to the nucleus. Also the nucleo-cytoplasmic trafficking of these patient-related SBDS proteins was disturbed. Further studies with a series of SBDS mutant proteins revealed that three distinct motifs determine the intracellular mobility of SBDS protein. A sumoylation motif in the C-terminal domain, that is lacking in patient SBDS proteins, was found to play a pivotal role in intracellular motility. Our structure-function analyses provide new insight into localization and motility of the SBDS protein, and show that patient-related mutant proteins are altered in their molecular properties, which may contribute to the clinical features observed in SDS patients.

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Related in: MedlinePlus

GFP-SBDS-FL intracellular transport is affected by ribosome and RNA processing inhibitors.(A) Representative GFP-SBDS-FL and (B) GFP-SBDS-FL-expressing cell in the presence of cyclohexamide (C) GFP-SBDS-FL expressing cell in the presence of actinomycinD for FRAP analysis prior to bleaching, at the moment of nuclear bleaching and 10 min post-bleach. (D) FRAP analysis showing the average relative nuclear/cytoplasmic GFP ratio for GFP-SBDS-FL (light grey curve), or GFP-SBDS-FL in the presence of actinomycinD (dark grey curve) or cyclohexamide (black curve). We analysed 8-9 cells in 2 independent experiments. (E) Representative GFP-SBDS-FL and (F) representative GFP-SBDS-FL expressing cell in the presence of cyclohexamide and (G) representative GFP-SBDS-FL expressing cell in the presence of actinomycinD for FLIP analysis prior to bleaching, at the moment of nuclear bleaching and 10 min post-bleach (H) FLIP analysis showing the average nuclear GFP intensity for GFP-SBDS-FL (lightgrey curve), GFP-SBDS-FL in the presence of cyclohexamide (black curve), GFP-SBDS-FL in the presence of actinomycinD. We analysed 7–12 cells in 3 independent experiments.
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pone-0020727-g005: GFP-SBDS-FL intracellular transport is affected by ribosome and RNA processing inhibitors.(A) Representative GFP-SBDS-FL and (B) GFP-SBDS-FL-expressing cell in the presence of cyclohexamide (C) GFP-SBDS-FL expressing cell in the presence of actinomycinD for FRAP analysis prior to bleaching, at the moment of nuclear bleaching and 10 min post-bleach. (D) FRAP analysis showing the average relative nuclear/cytoplasmic GFP ratio for GFP-SBDS-FL (light grey curve), or GFP-SBDS-FL in the presence of actinomycinD (dark grey curve) or cyclohexamide (black curve). We analysed 8-9 cells in 2 independent experiments. (E) Representative GFP-SBDS-FL and (F) representative GFP-SBDS-FL expressing cell in the presence of cyclohexamide and (G) representative GFP-SBDS-FL expressing cell in the presence of actinomycinD for FLIP analysis prior to bleaching, at the moment of nuclear bleaching and 10 min post-bleach (H) FLIP analysis showing the average nuclear GFP intensity for GFP-SBDS-FL (lightgrey curve), GFP-SBDS-FL in the presence of cyclohexamide (black curve), GFP-SBDS-FL in the presence of actinomycinD. We analysed 7–12 cells in 3 independent experiments.

Mentions: In the presence of actinomycin D, a transcription inhibitor known to affect ribosome production, we observed both an increase in GFP-SBDS-FL nuclear import as well as nuclear export in these cells (Fig. 5C/D/G/H). This suggests that SBDS-FL under steady-state conditions is retained in the nucleus in transcriptional or ribosomal protein complexes, which are disrupted upon actinomycin D treatment. Importantly, these data also provide indirect evidence that GFP-SBDS-FL protein is functional, since interference with ribosome and RNA processing affects mobility of the protein.


Altered intracellular localization and mobility of SBDS protein upon mutation in Shwachman-Diamond syndrome.

Orelio C, van der Sluis RM, Verkuijlen P, Nethe M, Hordijk PL, van den Berg TK, Kuijpers TW - PLoS ONE (2011)

GFP-SBDS-FL intracellular transport is affected by ribosome and RNA processing inhibitors.(A) Representative GFP-SBDS-FL and (B) GFP-SBDS-FL-expressing cell in the presence of cyclohexamide (C) GFP-SBDS-FL expressing cell in the presence of actinomycinD for FRAP analysis prior to bleaching, at the moment of nuclear bleaching and 10 min post-bleach. (D) FRAP analysis showing the average relative nuclear/cytoplasmic GFP ratio for GFP-SBDS-FL (light grey curve), or GFP-SBDS-FL in the presence of actinomycinD (dark grey curve) or cyclohexamide (black curve). We analysed 8-9 cells in 2 independent experiments. (E) Representative GFP-SBDS-FL and (F) representative GFP-SBDS-FL expressing cell in the presence of cyclohexamide and (G) representative GFP-SBDS-FL expressing cell in the presence of actinomycinD for FLIP analysis prior to bleaching, at the moment of nuclear bleaching and 10 min post-bleach (H) FLIP analysis showing the average nuclear GFP intensity for GFP-SBDS-FL (lightgrey curve), GFP-SBDS-FL in the presence of cyclohexamide (black curve), GFP-SBDS-FL in the presence of actinomycinD. We analysed 7–12 cells in 3 independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3113850&req=5

pone-0020727-g005: GFP-SBDS-FL intracellular transport is affected by ribosome and RNA processing inhibitors.(A) Representative GFP-SBDS-FL and (B) GFP-SBDS-FL-expressing cell in the presence of cyclohexamide (C) GFP-SBDS-FL expressing cell in the presence of actinomycinD for FRAP analysis prior to bleaching, at the moment of nuclear bleaching and 10 min post-bleach. (D) FRAP analysis showing the average relative nuclear/cytoplasmic GFP ratio for GFP-SBDS-FL (light grey curve), or GFP-SBDS-FL in the presence of actinomycinD (dark grey curve) or cyclohexamide (black curve). We analysed 8-9 cells in 2 independent experiments. (E) Representative GFP-SBDS-FL and (F) representative GFP-SBDS-FL expressing cell in the presence of cyclohexamide and (G) representative GFP-SBDS-FL expressing cell in the presence of actinomycinD for FLIP analysis prior to bleaching, at the moment of nuclear bleaching and 10 min post-bleach (H) FLIP analysis showing the average nuclear GFP intensity for GFP-SBDS-FL (lightgrey curve), GFP-SBDS-FL in the presence of cyclohexamide (black curve), GFP-SBDS-FL in the presence of actinomycinD. We analysed 7–12 cells in 3 independent experiments.
Mentions: In the presence of actinomycin D, a transcription inhibitor known to affect ribosome production, we observed both an increase in GFP-SBDS-FL nuclear import as well as nuclear export in these cells (Fig. 5C/D/G/H). This suggests that SBDS-FL under steady-state conditions is retained in the nucleus in transcriptional or ribosomal protein complexes, which are disrupted upon actinomycin D treatment. Importantly, these data also provide indirect evidence that GFP-SBDS-FL protein is functional, since interference with ribosome and RNA processing affects mobility of the protein.

Bottom Line: Further studies with a series of SBDS mutant proteins revealed that three distinct motifs determine the intracellular mobility of SBDS protein.A sumoylation motif in the C-terminal domain, that is lacking in patient SBDS proteins, was found to play a pivotal role in intracellular motility.Our structure-function analyses provide new insight into localization and motility of the SBDS protein, and show that patient-related mutant proteins are altered in their molecular properties, which may contribute to the clinical features observed in SDS patients.

View Article: PubMed Central - PubMed

Affiliation: Sanquin Research and Landsteiner Laboratory of the Academic Medical Center, Department of Blood Cell Research, University of Amsterdam, Amsterdam, The Netherlands.

ABSTRACT
Shwachman-Diamond Syndrome (SDS) is a rare inherited disease caused by mutations in the SBDS gene. Hematopoietic defects, exocrine pancreas dysfunction and short stature are the most prominent clinical features. To gain understanding of the molecular properties of the ubiquitously expressed SBDS protein, we examined its intracellular localization and mobility by live cell imaging techniques. We observed that SBDS full-length protein was localized in both the nucleus and cytoplasm, whereas patient-related truncated SBDS protein isoforms localize predominantly to the nucleus. Also the nucleo-cytoplasmic trafficking of these patient-related SBDS proteins was disturbed. Further studies with a series of SBDS mutant proteins revealed that three distinct motifs determine the intracellular mobility of SBDS protein. A sumoylation motif in the C-terminal domain, that is lacking in patient SBDS proteins, was found to play a pivotal role in intracellular motility. Our structure-function analyses provide new insight into localization and motility of the SBDS protein, and show that patient-related mutant proteins are altered in their molecular properties, which may contribute to the clinical features observed in SDS patients.

Show MeSH
Related in: MedlinePlus