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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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N-terminally truncated GFP-SBDS proteins localize to the cytoplasm and nucleus, but do have altered intracellular trafficking properties than GFP-SBDS-FL.(A) Schematic overview of the GFP-tagged SBDS constructs. (B) Western blot analysis shows that GFP-tagged SBDS proteins have the expected molecular sizes of 51 kDa, 49 kDa and 48 kDa for the GFP-SBDS Δ1–62, GFP-SBDS Δ1–75 and GFP-SBDS Δ1–85 respectively. (C) Representative picture of the GFP-SBDS Δ1–85 protein. Bottom panel show the GFP fluorescence intensity plots measured as indicated in the corresponding cells in the top panel. White bar represents 10 µm. (D) Average ratio of the nuclear/cytoplasmic GFP fluorescence intensity for the different GFP-tagged constructs. GFP-SBDSΔ1–62 and Δ1–75 are significantly more localized to the cytoplasm as compared to GFP-SBDS Δ1–85 (p<0.001). Error bar indicates s.e.m. (FL n = 42 cells, Δ1–62 n = 32 cells, Δ1–75 n = 28 cells, Δ1–85 n = 42 cells in 3–4 independent experiments). (E) FRAP analysis for nuclear import showing the average nuclear/cytoplasmic GFP ratio for GFP-GFP, GFP-SBDS-FL and GFP-SBDS Δ1–85. In total 6 cells per construct in 2 independent experiments were analysed. (F) FLIP analysis for nuclear export showing the average nuclear GFP intensity for GFP-GFP, GFP-SBDS-FL and GFP-SBDSΔ1–85. We analysed 7–11 cells per construct in 3 independent experiments.
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pone-0020727-g003: N-terminally truncated GFP-SBDS proteins localize to the cytoplasm and nucleus, but do have altered intracellular trafficking properties than GFP-SBDS-FL.(A) Schematic overview of the GFP-tagged SBDS constructs. (B) Western blot analysis shows that GFP-tagged SBDS proteins have the expected molecular sizes of 51 kDa, 49 kDa and 48 kDa for the GFP-SBDS Δ1–62, GFP-SBDS Δ1–75 and GFP-SBDS Δ1–85 respectively. (C) Representative picture of the GFP-SBDS Δ1–85 protein. Bottom panel show the GFP fluorescence intensity plots measured as indicated in the corresponding cells in the top panel. White bar represents 10 µm. (D) Average ratio of the nuclear/cytoplasmic GFP fluorescence intensity for the different GFP-tagged constructs. GFP-SBDSΔ1–62 and Δ1–75 are significantly more localized to the cytoplasm as compared to GFP-SBDS Δ1–85 (p<0.001). Error bar indicates s.e.m. (FL n = 42 cells, Δ1–62 n = 32 cells, Δ1–75 n = 28 cells, Δ1–85 n = 42 cells in 3–4 independent experiments). (E) FRAP analysis for nuclear import showing the average nuclear/cytoplasmic GFP ratio for GFP-GFP, GFP-SBDS-FL and GFP-SBDS Δ1–85. In total 6 cells per construct in 2 independent experiments were analysed. (F) FLIP analysis for nuclear export showing the average nuclear GFP intensity for GFP-GFP, GFP-SBDS-FL and GFP-SBDSΔ1–85. We analysed 7–11 cells per construct in 3 independent experiments.

Mentions: To exclude the possibility that nuclear import and export of GFP-SBDS-FL was hampered due to molecular size constraints, we generated a GFP-GFP fusion protein which has a comparable molecular weight as GFP-SBDS-FL. As shown in Figure 3E/F, the GFP-GFP fusion protein was rapidly imported into and exported from the nucleus, although import/export kinetics were slower than the single free GFP (42% recovery in 10 minutes for nuclear import of GFP-GFP). Furthermore, to exclude the possibility that either SBDS protein expression levels or the position of the GFP-tag disturbs localization and mobility to great extent, we examined individual cells with different GFP-SBDS protein expression levels. We observed that expression levels did not affect GFP-SBDS protein isoform cellular behavior.


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)

N-terminally truncated GFP-SBDS proteins localize to the cytoplasm and nucleus, but do have altered intracellular trafficking properties than GFP-SBDS-FL.(A) Schematic overview of the GFP-tagged SBDS constructs. (B) Western blot analysis shows that GFP-tagged SBDS proteins have the expected molecular sizes of 51 kDa, 49 kDa and 48 kDa for the GFP-SBDS Δ1–62, GFP-SBDS Δ1–75 and GFP-SBDS Δ1–85 respectively. (C) Representative picture of the GFP-SBDS Δ1–85 protein. Bottom panel show the GFP fluorescence intensity plots measured as indicated in the corresponding cells in the top panel. White bar represents 10 µm. (D) Average ratio of the nuclear/cytoplasmic GFP fluorescence intensity for the different GFP-tagged constructs. GFP-SBDSΔ1–62 and Δ1–75 are significantly more localized to the cytoplasm as compared to GFP-SBDS Δ1–85 (p<0.001). Error bar indicates s.e.m. (FL n = 42 cells, Δ1–62 n = 32 cells, Δ1–75 n = 28 cells, Δ1–85 n = 42 cells in 3–4 independent experiments). (E) FRAP analysis for nuclear import showing the average nuclear/cytoplasmic GFP ratio for GFP-GFP, GFP-SBDS-FL and GFP-SBDS Δ1–85. In total 6 cells per construct in 2 independent experiments were analysed. (F) FLIP analysis for nuclear export showing the average nuclear GFP intensity for GFP-GFP, GFP-SBDS-FL and GFP-SBDSΔ1–85. We analysed 7–11 cells per construct in 3 independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3113850&req=5

pone-0020727-g003: N-terminally truncated GFP-SBDS proteins localize to the cytoplasm and nucleus, but do have altered intracellular trafficking properties than GFP-SBDS-FL.(A) Schematic overview of the GFP-tagged SBDS constructs. (B) Western blot analysis shows that GFP-tagged SBDS proteins have the expected molecular sizes of 51 kDa, 49 kDa and 48 kDa for the GFP-SBDS Δ1–62, GFP-SBDS Δ1–75 and GFP-SBDS Δ1–85 respectively. (C) Representative picture of the GFP-SBDS Δ1–85 protein. Bottom panel show the GFP fluorescence intensity plots measured as indicated in the corresponding cells in the top panel. White bar represents 10 µm. (D) Average ratio of the nuclear/cytoplasmic GFP fluorescence intensity for the different GFP-tagged constructs. GFP-SBDSΔ1–62 and Δ1–75 are significantly more localized to the cytoplasm as compared to GFP-SBDS Δ1–85 (p<0.001). Error bar indicates s.e.m. (FL n = 42 cells, Δ1–62 n = 32 cells, Δ1–75 n = 28 cells, Δ1–85 n = 42 cells in 3–4 independent experiments). (E) FRAP analysis for nuclear import showing the average nuclear/cytoplasmic GFP ratio for GFP-GFP, GFP-SBDS-FL and GFP-SBDS Δ1–85. In total 6 cells per construct in 2 independent experiments were analysed. (F) FLIP analysis for nuclear export showing the average nuclear GFP intensity for GFP-GFP, GFP-SBDS-FL and GFP-SBDSΔ1–85. We analysed 7–11 cells per construct in 3 independent experiments.
Mentions: To exclude the possibility that nuclear import and export of GFP-SBDS-FL was hampered due to molecular size constraints, we generated a GFP-GFP fusion protein which has a comparable molecular weight as GFP-SBDS-FL. As shown in Figure 3E/F, the GFP-GFP fusion protein was rapidly imported into and exported from the nucleus, although import/export kinetics were slower than the single free GFP (42% recovery in 10 minutes for nuclear import of GFP-GFP). Furthermore, to exclude the possibility that either SBDS protein expression levels or the position of the GFP-tag disturbs localization and mobility to great extent, we examined individual cells with different GFP-SBDS protein expression levels. We observed that expression levels did not affect GFP-SBDS protein isoform cellular behavior.

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