Lecture_11_Jan_29_

Lecture_11_Jan_29_ - Cytoplasm-Nucleus Transport reading...

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1 Cytoplasm-Nucleus Transport reading assignment: 704-712
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2 Cytoplasm-Nucleus Transport What is the property of the nuclear membrane e nuclear envelope)? (i.e. nuclear envelope)? How is a cargo protein transported into and out of the nucleus? What is the machinery mediating nuclear transport?
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3 Nuclear transport is abolished after the permeabilization of the plasma membrane Digitonin makes holes selectively on the plasma membrane and leaves the intracellular membranes intact Cytoplasmic content is lost but not most nuclear proteins (since nuclear membrane remains intact). 1. Add GFP-NLS fusion (>60 kDa) 2. Incubate o allow transport to occur) No nuclear transport can be detected (to allow transport to occur) 3. wash to remove cytoplasmic fusion (to exmaine the nuclear import) (as shown by the lack of green fluorescence in the nucleus) Cytosolic factors are required for the nuclear transport
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4 Nuclear transport of permeabilized cells can be restored after the addition of cytosol extracts (containing cytosolic factors) Digitonin makes holes selectively on e plasma membrane and leaves the plasma membrane and leaves the intracellular membranes intact Cytoplasmic content is lost but not most nuclear proteins (since nuclear membrane remains intact). GFP-NLS 1. Add GFP-NLS fusion (>60 kDa) 2. Incubate in the presence of cytosol extract (to allow transport to occur) 3. wash to remove cytoplasmic fusion (to exmaine the nuclear import) nuclear transport resumes The cytosol extracts provide the missing cytosolic factors.
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5 NLS-GFP (the transport cargo)
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Lecture_11_Jan_29_ - Cytoplasm-Nucleus Transport reading...

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