1. From Cytosol to organelles---post-translational translocation The big picture :

2. From Cytosol to the nucleus The road map: nucleus structure 1)The envelope: this is the barrier that separates N from cytosolic space. Inner and outer membrane: the outer membrane is connected with ER, perinuclear space connected with ER lumen. 2) Nuclear pores Very large protein complex of more than 100 different proteins forms a basket-like structure through the double membranes

4. The pore complex in plant cells is less well studied but it has been show to contain glycoproteins (probed by lectins that bind sugar groups) 3) Diffusion and “active” transport through the pore: Small molecules are freely diffusing back and forth but molecules larger than 60 KD need active transport and require signal sequence for getting in. 4) Two way traffic! Proteins get in and RNAs get out

5. 2. The signal sequence and the mechanism of import 1)Nuclear localization signal (NLS) Classical experiments using frog oocytes and microinjection: gold particles coated with a nuclear protein—dissecting the signal sequence/domain Head tail tail Head

6. Many procedures can be used to study the signal sequences that are responsible for nuclear import: in vitro experiments using isolated cells or nucleus and in vivo experiments using transgenic plants or transiently transformed cells—monitoring the presence of your protein of interest (by labeling them with fluorescence or other detectable signal). Conclusion: a) The nuclear localization signal (NLS) often consists of two patches of positively charged amino acids (4 or more residues in each patch). This pattern is not very strictly enforced. b) Such sequence can direct other proteins or even gold particles into the nucleus---no photo ID required. C) Not cleaved! XXXX(R/K)nXXXXXXXX (R/K)n

7. 2) Receptors for NLS: In vitro assay of nuclear transport: permeabilized animal culture cells---plus the labeled protein---does not go into the nucleus even when NLS is present---plus ATP and cell lysate (cytoplasmic components)---goes to nucleus: Conclusion: ATP and cytoplasmic proteins/factors are required for import. Using this assay, the soluble factors were purified as importin alpha and beta and a small G protein called Ran. The importins form dimeric complex that recognize NLS and bind the target protein. IN the presence of ATP, importin beta interacts with nuclear pore complex (NPC) and they get into the nucleus. What is Ran doing? Ran is shown to facilitate the dissociation of cargo from importin complex and the export of importin beta for the shuttling process. The proteins GAP and GEF are GTPase activation protein (GAP) and guanine nucleotide exchange factor (GEF). They regulate the activity of Ran.

9. 3) Regulation of the import traffic: Control of protein import provides another mechanism for cell regulation—protein is produced but may not be imported into the nucleus—who has the priority to enter? This control can be performed by many possible ways such as protein phosphorylation or dephosphorylation, protein complex formation, and so on. These processes can be tightly controled by presence of signals including hormones or environmental cues. In animal cells, a classical example is steroid receptor import. In plant cells, light-regulated protein import—COP1 repressor protein is present in both cytosol and nucleus in light but only in nucleus in the dark.

10. NLS hsp90 steroids NLS hsp90 NLS Gene activation

11. 3. Nuclear Export 1)What to be exported? mRNA as an example. mRNA in the nucleus is packaged by proteins forming ribonucleoproteins (RNPs). 2) Nuclear export signal (NES): is in the proteins that associate with the mRNA. The details of such signals are not clear because several different sequences have been shown to serve as NES in different proteins—more diverse than the NLS. 3) The receptors: exportins The receptors for the NES sequences are called exportins (vs importins for import). The cycle of exportin action is quite similar to the importin cycle and also involves Ran, the small G protein.

12. Comparing the import and export: a)some components like Ran are shared. b)They also share the same pores (one pore can handle two way traffic) c)But “tickets” and receptors are different

13. Other export activities: the mechanisms for other types of RNAs are less understood