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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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SDS-patient SBDS proteins are localized to the nucleus.(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 59 kDa, 36 kDa, 39 kDa and 54 kDa for the GFP-SBDS-FL, GFP-SBDS-K62, GFP-SBDS-C84 and GFP-SBDS-R218 respectively. (C) Representative pictures of the intracellular localization of the GFP-tagged SBDS proteins. Bottom panel shows the GFP fluorescence intensity plots measured as indicated in the corresponding cells in the top panel. (D) Average ratio of the nuclear/cytoplasmic GFP fluorescence intensity for the different GFP-tagged constructs Asterisk indicates that the localization of the GFP-SBDS-FL is statistically significant different (p<0.001) from the SDS-patient GFP-SBDS proteins. Error bar indicates s.e.m.(FL n = 42 cells, K62 n = 52 cells, C84 n = 37 cells, R218 n = 33 cells analysed in 3–5 independent experiments).
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pone-0020727-g001: SDS-patient SBDS proteins are localized to the nucleus.(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 59 kDa, 36 kDa, 39 kDa and 54 kDa for the GFP-SBDS-FL, GFP-SBDS-K62, GFP-SBDS-C84 and GFP-SBDS-R218 respectively. (C) Representative pictures of the intracellular localization of the GFP-tagged SBDS proteins. Bottom panel shows the GFP fluorescence intensity plots measured as indicated in the corresponding cells in the top panel. (D) Average ratio of the nuclear/cytoplasmic GFP fluorescence intensity for the different GFP-tagged constructs Asterisk indicates that the localization of the GFP-SBDS-FL is statistically significant different (p<0.001) from the SDS-patient GFP-SBDS proteins. Error bar indicates s.e.m.(FL n = 42 cells, K62 n = 52 cells, C84 n = 37 cells, R218 n = 33 cells analysed in 3–5 independent experiments).

Mentions: To gain more insight into the cellular and molecular function of SBDS, we examined the subcellular localization of the full length GFP- and HA-tagged SBDS-FL protein. Also, we introduced translational stopcodons at K62, at C84 or at R218 to mimic SDS patient SBDS truncated proteins (Fig. 1A). Western blot analysis of transiently transfected HeLa cells with these GFP-tagged or HA-tagged SBDS constructs showed that these proteins are expressed and have the expected molecular sizes (Fig. 1B; Suppl. Fig. S1).


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)

SDS-patient SBDS proteins are localized to the nucleus.(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 59 kDa, 36 kDa, 39 kDa and 54 kDa for the GFP-SBDS-FL, GFP-SBDS-K62, GFP-SBDS-C84 and GFP-SBDS-R218 respectively. (C) Representative pictures of the intracellular localization of the GFP-tagged SBDS proteins. Bottom panel shows the GFP fluorescence intensity plots measured as indicated in the corresponding cells in the top panel. (D) Average ratio of the nuclear/cytoplasmic GFP fluorescence intensity for the different GFP-tagged constructs Asterisk indicates that the localization of the GFP-SBDS-FL is statistically significant different (p<0.001) from the SDS-patient GFP-SBDS proteins. Error bar indicates s.e.m.(FL n = 42 cells, K62 n = 52 cells, C84 n = 37 cells, R218 n = 33 cells analysed in 3–5 independent experiments).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3113850&req=5

pone-0020727-g001: SDS-patient SBDS proteins are localized to the nucleus.(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 59 kDa, 36 kDa, 39 kDa and 54 kDa for the GFP-SBDS-FL, GFP-SBDS-K62, GFP-SBDS-C84 and GFP-SBDS-R218 respectively. (C) Representative pictures of the intracellular localization of the GFP-tagged SBDS proteins. Bottom panel shows the GFP fluorescence intensity plots measured as indicated in the corresponding cells in the top panel. (D) Average ratio of the nuclear/cytoplasmic GFP fluorescence intensity for the different GFP-tagged constructs Asterisk indicates that the localization of the GFP-SBDS-FL is statistically significant different (p<0.001) from the SDS-patient GFP-SBDS proteins. Error bar indicates s.e.m.(FL n = 42 cells, K62 n = 52 cells, C84 n = 37 cells, R218 n = 33 cells analysed in 3–5 independent experiments).
Mentions: To gain more insight into the cellular and molecular function of SBDS, we examined the subcellular localization of the full length GFP- and HA-tagged SBDS-FL protein. Also, we introduced translational stopcodons at K62, at C84 or at R218 to mimic SDS patient SBDS truncated proteins (Fig. 1A). Western blot analysis of transiently transfected HeLa cells with these GFP-tagged or HA-tagged SBDS constructs showed that these proteins are expressed and have the expected molecular sizes (Fig. 1B; Suppl. Fig. S1).

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