An improved genetic system for detection and analysis of protein nuclear import signals

Marshall, Kris; Zhang, Zhiying; Curran, Jennifer; Derbyshire, Stephanie; Mymryk, Joe S.

doi:10.1186/1471-2199-8-6

Cited by 14 publications

(19 citation statements)

References 20 publications

(34 reference statements)

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“…We fused a series of sequences spanning the entire Fra ICD to a transcription factor consisting of a DNA-binding domain from the bacterial transcription factor LexA (mutLexA DBD) and an activation domain from the yeast transcription factor Gal4 (Gal4 AD). The mutLexA DBD that we used in these experiments has mutations that abolish its intrinsic ability to enter the nucleus (Rhee et al, 2000; Marshall et al, 2007) and the Gal4 AD does not localize to the nucleus (Silver et al, 1988). We expressed these fusion proteins in ADE2 reporter yeast and identified three different regions of the Fra ICD that are sufficient to confer nuclear localization (Figure S1C).…”

Section: Resultsmentioning

confidence: 99%

The Intracellular Domain of the Frazzled/DCC Receptor Is a Transcription Factor Required for Commissural Axon Guidance

Neuhaus-Follini

Bashaw

2015

Neuron

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Summary In commissural neurons of Drosophila, the conserved Frazzled (Fra)/Deleted in Colorectal Cancer (DCC) receptor promotes midline axon crossing by signaling locally in response to Netrin and by inducing transcription of commissureless (comm), an antagonist of Slit-Roundabout midline repulsion, through an unknown mechanism. Here, we show that Fra is cleaved to release its intracellular domain (ICD), which shuttles between the cytoplasm and the nucleus, where it functions as a transcriptional activator. Rescue and gain-of-function experiments demonstrate that the Fra ICD is sufficient to regulate comm expression and that both γ-secretase proteolysis of Fra and Fra's function as a transcriptional activator are required for its ability to regulate comm in vivo. Our data uncover an unexpected role for the Fra ICD as a transcription factor whose activity regulates the responsiveness of commissural axons at the midline and raise the possibility that nuclear signaling may be a common output of axon guidance receptors.

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Section: Resultsmentioning

confidence: 99%

The Intracellular Domain of the Frazzled/DCC Receptor Is a Transcription Factor Required for Commissural Axon Guidance

Neuhaus-Follini

Bashaw

2015

Neuron

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“…For nuclear localization trap assays (Marshall et al, 2007), full-length MGG_03307 was expressed in the yeast strain L40 (MATa, trp1, leu2, his3, LYS2∷lex A-HIS3, URA3∷lex A-lacZ) as a fusion with the mLexA-MBP-Gal4AD polypeptide. To this end, the coding sequence plus a portion of the 3’-flaking region of MGG_03307 (cds-MGG_03307: +1; +1500) was amplified with the CVNHcds.fw EcoRI and CVNHcds.rv SalI primers (Table S1) and inserted into the Eco RI/ Sal I digsted pNIA-CEN-MBP vector (Marshall et al, 2007).…”

Section: Methodsmentioning

confidence: 99%

“…To this end, the coding sequence plus a portion of the 3’-flaking region of MGG_03307 (cds-MGG_03307: +1; +1500) was amplified with the CVNHcds.fw EcoRI and CVNHcds.rv SalI primers (Table S1) and inserted into the Eco RI/ Sal I digsted pNIA-CEN-MBP vector (Marshall et al, 2007). The expression of two reporter genes (HIS3 and LacZ) was analyzed using previously described methods (Walhout AJ et al 2001, Methods 24: 297–306) with the empty vector as a negative control.…”

Section: Methodsmentioning

confidence: 99%

Structure-Function Analysis of a CVNH-LysM Lectin Expressed during Plant Infection by the Rice Blast Fungus Magnaporthe oryzae

et al. 2011

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Summary The rice blast fungus Magnaporthe oryzae’s genome encodes a hypothetical protein (MGG_03307) containing a type III CVNH lectin, in which a LysM domain is inserted between individual repeats of a single CVNH domain. At present, no structural or ligand binding data is available for any type III CVNH and functional studies in natural source organisms are scarce. Here, we report NMR solution structure and functional data on MGG_03307. The structure of the CVNH/LysM module revealed that intact and functionally competent CVNH and LysM domains are present. Using NMR titrations, carbohydrate specificities for both domains were determined, and it was found that each domain behaves as an isolated unit without any inter-domain communication. Furthermore, live-cell imaging revealed a predominant localization of MGG_03307 within the appressorium, the specialized fungal cell for gaining entry into rice tissue. Our results suggest that MGG_03307 plays a role in the early stages of plant infection.

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“…Fragment 225-297 contains clusters of positively charged amino acid residues that fit the consensus sequence of a classical NLS (37). To test whether this segment promotes transport into the nucleus, we generated GFP-hsc70(225-297)-␤-galactosidase, a reporter protein that contains GFP-hsc70(225-297) fused to ␤-galactosidase.…”

Section: Residues 225 To 297 Are Necessary But Not Sufficient Formentioning

confidence: 99%

Nucleolar Targeting of the Chaperone Hsc70 Is Regulated by Stress, Cell Signaling, and a Composite Targeting Signal Which Is Controlled by Autoinhibition

Bański

Mahboubi

Kodiha

et al. 2010

Journal of Biological Chemistry

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Hsc70s are constitutively synthesized members of the 70-kDa chaperone family; they are essential for viability and conserved among all organisms. When eukaryotic cells recover from stress, hsc70s accumulate in nucleoli by an unknown mechanism. Our studies were undertaken to characterize the signaling events and the targeting sequence required to concentrate hsc70 in the nucleoli of human cells. Here, we show that pharmacological inhibitors of phosphatidylinositol (PI) 3-kinase and MEK kinases as well as protein-tyrosine phosphatases abolished the stress-dependent nucleolar accumulation of hsc70. Furthermore, to identify the hsc70 nucleolar targeting sequence, green fluorescent protein-tagged fusion proteins with defined segments of hsc70 were generated and their subcellular distribution was analyzed in growing cells. These studies demonstrated that residues 225 to 297 serve as a heat-inducible nucleolar targeting signal. This segment directs green fluorescent protein to nucleoli in response to stress, but fails to do so under nonstress conditions. Fine mapping of the nucleolar targeting signal revealed that it has two separable functions. First, residues 225 to 262 direct reporter proteins constitutively to nucleoli, even without stress. Second, segment 263 to 287 functions as an autoinhibitory element that prevents hsc70 from concentrating in nucleoli when cells are not stressed. Taken together, PI 3-kinase and MEK kinase signaling as well as tyrosine dephosphorylation are essential for the accumulation of hsc70 in nucleoli of stressed cells. This process relies on a stress-dependent composite targeting signal that combines multiple functions.Heat shock cognate proteins 70 (hsc70) 4 are members of the 70-kDa family of chaperones, which are conserved among all organisms. Unlike cytoplasmic hsp70s, hsc70s are constitutively synthesized and essential for cell survival (1). Hsp/hsc70s are involved in a large number of cellular processes: they contribute to the proper folding of nascent polypeptides, refolding of denatured proteins, and targeting of damaged proteins to the proteasome (reviewed in Refs. 2-4). Hsp/hsc70s are essential for protein sorting to organelles and play a protective role in many human diseases and pathophysiologies, including Alzheimer and Huntington disease or ischemia of the heart and brain (reviewed in Refs. 5 and 6). Furthermore, defects in chaperone function have been linked to human disease and aging (5, 7-9).Members of the hsp/hsc70 family are organized into three domains; the 44-kDa N-terminal ATPase domain is followed by a protein binding segment of 18 kDa and a 10-kDa variable domain at the C-terminal end (reviewed in Ref. 3). Under normal growth conditions, hsc70s shuttle between the nucleus and the cytoplasm; however, hsc70s shuttling is inhibited upon stress (10). Following heat shock, hsc70s initially accumulate in the nucleoplasm, where they associate with chaperone substrates that require refolding (10, 11). When cells recover from stress, hsc70s transiently concentrate in nuc...

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An improved genetic system for detection and analysis of protein nuclear import signals

Cited by 14 publications

References 20 publications

The Intracellular Domain of the Frazzled/DCC Receptor Is a Transcription Factor Required for Commissural Axon Guidance

The Intracellular Domain of the Frazzled/DCC Receptor Is a Transcription Factor Required for Commissural Axon Guidance

Structure-Function Analysis of a CVNH-LysM Lectin Expressed during Plant Infection by the Rice Blast Fungus Magnaporthe oryzae

Nucleolar Targeting of the Chaperone Hsc70 Is Regulated by Stress, Cell Signaling, and a Composite Targeting Signal Which Is Controlled by Autoinhibition

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