Nucleo cytoplasmic trafficking
- General Overview
- Lab interests
- A. Biogenesis of UsnRNPs
- 1. Cap Hypermethylation
- 2.Recognition of the core UsnRNP by SPN1
- 3. Import properties of SPN1 and Binding to Importin ß
- 4. Recycling of SPN1
B. Import of linker Histones
A hallmark of eukaryotes is the compartmentalization of the cell. One of the gained compartments, the nucleus enables the cell to separate nuclear transcription from cytoplasmic translation. This offers a multitude of additional control mechanisms for these processes. The downside of the spatial separation is the need to transport macromolecules, like proteins and ribonucleic acid (RNA) across this membrane (Fig.1)
. This process is essential for cellular function and requires an active nuclear–cytoplasmic transport machinery. This machinery is based on soluble transport receptors, which recognize and bind cargo in one compartment, mediate its transport through so called nuclear pore complexes (NPCs) which are embedded in the double membrane of the nucleus and release the cargo in the target compartment after traverse.
Fig.1. Exchange of molecules across the nuclear envelope.
mRNA has to be exported into the cytoplasm in order to be translated, tRNA and ribosomes have to be exported as well, whereas regulatory proteins as well as histones and ribosomal proteins have to be imported into the nucleus.
The majority of nucler transport receptors belong to the Importin ß superfamily of receptors or ß-karyopherins, named after the first receptor identified. To specify the direction of transport karyopherins have been separated in importins and exportins. All members of the Importin ß superfamily share a common structural feature, termed HEAT repeats, first identified in Huntingtin, elongation factor 3 (EF3), protein phosphatase 2A (PP2A), and the yeast PI3-kinase TOR1. A HEAT repeat consists of two antiparallel alpha-helices separated by a short loop (Fig.2A.)
. The N-terminal helix is denoted A-helix and is generally located at the outside of the protein, wheras the second helix, the B-helix is directed to the inner surface of the protein. The arrangement of multiple HEAT repeats leads to a superhelical structure as shown for importin ß composed of 19 HEAT-repeats (Fig.2B.)
Fig. 2. Structural arrangment of the Importin ß superfamily.
A. HEAT-repeat structure. B.Overall Arrangement of HEAT repeats in Importin ß.
[Click for a larger image]
This superhelical arrangement allows for a high dergree of flexibility, enabling the interaction with a wide range of divergent protein (Fig.3.)
. The shape of the superhelix depicted in green is caused upon binding to an N-terminal alpha helical structure od a protein binding along the C-terminal B-helices within importin ß along the central axis. The C-terminal core domain of the bound protein is most likely located on the top of importin ß. In the other form of importin ß depicted in purple, the cargo is bound to the B-helices in the central region with the main body of the bound molecule presumably lying on the the left. The two conformatins of importin ß differ in respect to overall hight and diameter.
Fig.3. Flexibility of Importin ß.
The mode of interaction with the caro causes different conformations of importin ß, which show differences in length and diameter.
Besides a high degree of structural similarity, all members of the importin ß superfamily share the following binding properties:
-Ran binding in the N-terminal region
Recognition of cargo proteins
Due to the fact that the nucleus is degraded and rebuilt every cell cycle, proteins with prolonged half life have to be imported in the nucleus after every re-formation of the nuclear envelope. Others have to shuttle between the compartments. Thus the recognition requires a permanent surface exposed signal which is not cleaved off after translocation like for other transport pathways e.g. in the ER and thus may be an integral part the protein. This is a general feature of proteins trafficking the nuclear envelope.
The first localization signal identified for the import and export pathway have been named classical NLS (nuclear localisation signal) and NES (nuclear export signal), repectively (Fig.4.).
Fig.4. Nuclear Transport signals.
The classical NLS may be monopartite - with 5 out of 7 residues basic - or bipartite. The second part is 10-12 residues distant composed of short stretch of basic residues. The classical NES in contrast exhibits a series of hydrophobic residues separated by 1-3 residues.
They are recognized by CRM1 for NESs or an importin ß-dependent pathway for NLSs. The interaction of NLS bearing cargo with importin ß is indirect and requires a bridging or „adapter“ molecule, importin alpha.
Subsequently, further general import signals have been identified, like the PY-motif for cargoes imported by Transportin, another member of the importin ß superfamily.
Directionality of transport receptors
Fig.5. Directionality of transport is mediated by the small GTPase Ran.
The Ran is loaded with GTP by the guanine nucleotide exchange factor RCC1 and its intrinsically low hydrolysis rate is triggered by the GTPase activating protein RanGAP and even more accelerated by binding of RanBPs (Binding Proteins).
In order to obtain effcient transport, the cargo has to be bound by the receptor in one compartment and released in the other compartment. This is accopmlished by the small GTPase Ran. Ran exists in two flavours, the GTP bound form and the GDP bound form. Ran exhibits a low intrinsic hydrolysis activity and requires additional factors which aid in accelerating the hydrolysis. These proteins so called RanGAPs (GTPase Activating Proteins) reside in the cytoplasmic compartment either in a free state or bound to cytoplasmic extensions of the NPC. Moreover the cell contains Ran binding proteins (RanBPs) further increasing the hydrolysis rate (Fig.5.). Once Ran has hydrolysed the GTP to GDP and inorganic phospahte, Ran has to be reverted to the GTP bound form by Ran gunaine nucleotide exchange factor RCC1 (regulator of chromosome condensation 1) into the GTP bound form. RCC1 is localized exclusively in the nuclear compartment. Thus Ran in the GTP bound form is predominantly in the nucleus, whereas the GDP bound form is mainly restricted to the cytoplasmic compartment. This steep gradient across the nuclear membrane is a prerequisite for efficient nuclear trafficking. RanGTP binds to receptors in the nucleus and traverses to the cytoplasm, wher the hydrolysis of GTP by RanGAP triggers its release from the receptor. For a recharging with GTP it is actively transported back into the nucleus by NTF2 (nuclear transport factor 2) (Fig.6.)
Fig.6. A steep gradient across the nuclear envelope drives the directionality of transport.
RanGTP is required for the tansfer of receptors belonging to the Importin ß superfamily into the cytoplasm. After hydrolysis and dissociation from receptors RanGDP itself is shuttled back into the nucleus by NTF2.
The established gradient and the binding properties of Ran are also the cause for the directed transport of cargoes by the different receptors. Whereas importins bind their cargo only in the absence of Ran, exportins require the presence of Ran for cargo binding. As mentioned above all receptors bind to Ran in the GTP bound form. Thus importins bind their cargo in the cytoplasm traverse through the NPC, RanGTP binding triggers the release of the cargo and the RanGTP- bound importins are destined for export back into the cytoplasm (Fig.7.). On the contrary exportins have the opposite properties. They bind the cargo only when RanGTP is present, ergo in the nucleus and release it in the cytoplam retuning in the nucleus in an empty state, where upon binding to RanGTP an new cycle begins.
Fig.7. Transport cycle for Importins and Exportins.
. Importins bind their cargo in the cytoplasm and deliver it to the nucleus. Upon binding of RanGTP the cargo is released and the importin is shuttled back into the cytoplasm. In contrast, exportins only bind their cargo in the presence of RanGTP, thus in the nucleus and release it in the cytoplasmic compartment after hydrolysis of RanGTP.
The variability of cargoes has led to a huge variety of localisation signals. In order to fulfill the requirements to achive the recognition of all the different signals karyopherins have evolved the abovementioned flexibility and often binding regions with a broad albeit specific recognition capability. Moreover they may contain more than one binding region or use adaptors to bind cargoes. Adaptors themselves bear regions mediating specific binding to the receptor and a specific binding region for the cargo, thus bridging the interaction between cargo and receptor, increasing the number of localisation signals even more. Importin ß is such a receptor that has multiple ways of recognising its cargoes. Either it recognizes the cargo directly or uses the help of such adaptor molecules to achieve its task. Two adaptors Importin alpha and Snurportin 1 (SPN1) have been extensively studied. Besides the diverging cargo binding regions – one binds proteins, the other specific cap structures of UsnRNAs (see below). Both share a similar N-terminal region for Importin ß binding, the IBB domain. Interestingly both adaptors require exportins for theirrecycling to the cytoplasm. Whereas SPN1 is exported by a general export receptor CRM1 (Chromosome region maintenace 1; also making use of adaptors to achieve its task), Importin alpha is exported by a specific exportin, CAS (Cellular Apoptosis Susceptibility gene product)
A third way of forming import complexes requires the interaction of importin ß with a second receptor importin 7, a so called two receptor pathway required for the import of linker histones.
Biogenesis of UsnRNPs
In eukaryotic cells mRNAs are generally transcribed as pre-mRNAs in which the information for the protein sequence is contained in so called exons and intervening non-coding regions, the introns. Thus, the newly synthesized pre-mRNA is composed of an array of exons and introns. In order to obtain meaningful genetic information leading to functional protein, the introns have to be removed, a process occuring before export of the mRNA to the cytoplasm where translation by the ribosome takes place. The excision of an intron and fusion of the adjacent exons is achieved by the spliceosome, a supramolecular, highly dynamic, ribonucleoprotein (RNP) complex.
Essential components of the major spliceosome are the five Uridyl-rich small nuclear RNPs (UsnRNPs) U1, U2, U4, U5 and U6 and several non-snRNP proteins. Each UsnRNP is composed of a specific UsnRNA and a set of seven common proteins, the Sm proteins, for U1, U2, U4, U5 or highly homologous proteins to those seven, the Lsm proteins, for U6. Additionally each UsnRNP acquires a subset of particle specific protein.
Fig.8. Biogenesis of UsnRNPs.
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The biogenesis of spliceosomal UsnRNPs in higher eukaryotes requires a cytoplasmic maturation step (Fig.8). Thus, after transcription and initial processing within the nucleus the snRNAs U1, U2, U4 and U5 are exported to the cytoplasm in an m7G-Cap dependent manner by the CRM1 dependent pathway as well as the proteins PHAX, CBP20 and 80 as mediators. In the cytoplasm these UsnRNAs specifically associate with seven Sm-proteins that form a doughnut-shaped UsnRNP core structure. This assembly, its formation mediated by the SMN complex, is a prerequisite for the hypermethylation of the m7G-cap to the 2,2,7-trimethylguanosine (m3G)-cap (Step 1). Snurportin1 specifically recognises this m3G-cap and in concert with other import factors facilitates the import of core UsnRNPs into the nucleus (Step 2/3). Here the complex disassembles in an ordered fashion and SPN1 is transported back into the cytoplasm in a CRM1/RanGTP dependent manner (Step 4)
For a more detailed information on the biogenesis of UsnRNPs and the underlying transport processes see:
Dickmanns, A. (2009). Import and Export of UsnRNPs. Nuclear Transport; Edited by Ralph H. Kehlenbach, Landes Bioscience
Dickmanns, A. and Ficner, R. (2005). Role of the 5’-cap in the biogenesis of spliceosomal snRNPs. Topics in Current Genetics 12, 179-204.
Recent research has focussed on the structural requirements for the interaction of the following steps in the biogenesis (numbers according to the numbers in Fig.8.):
1. UsnRNA 5'-cap hypermethylation by TGS1
The trimethylguanosine synthase TGS1 comprises a region resembling the canonical methyltransferase domain for substrate and ligand (SAM) binding. Structure determination of this domain revealed a fold structurally similar to the core domain of methyltrasferases, but activity tests showed no activity (Fig.9. right). Stepwise addition of residues at the N-terminus led to active forms of TGS1. Structural analysis of the active form revealed an additional N-terminal domain, its correct formation essential for binding to both ligand and substrate (Fig.9.).
Fig.9. TGS1 requires an N-terminal domain additionally to the MTase domain for activity.
Monecke, T., Dickmanns, A., and Ficner, R. (2009). Structural basis for m7G-cap hypermethylation of small nuclear, small nucleolar and telomerase RNA by the dimethyltransferase TGS1.Nucleic Acids Res. 37, 3865-3877.
Monecke,T., Dickmanns, A., Strasser A., and Ficner R. (2009). Structure Analysis of the conserved Methyltransferase Domain of Human Trimethylguanosine Synthase TGS1.
Acta Cryst. D65, 332-338. [Abstract]; PDB:[3EGI]
2. Recognition of the core UsnRNP by SPN1
Snurportin1 acts as an adaptor between cargo – teh core UsnRNP – and the import receptor importin ß. The N-terminal IBB domain interactts with Importin ß whereas the c-terminal region binding to –UsnRNPs specifically regognizes the hypermethylated cap structure (Fig.10.)
. Interestingly, and in stark contrast to other cap binding proteins, importin ß binds to the first two nucleotides of the RNA which are arranged in a stacked conformation.
Fig.10. Import adaptor Snurportin1
Left: SPN1 domain architecture and overall structure of the cap binding domain. Right: The trimethylated cap and the first nucleotide are both required for binding to SPN1. The two nucleotides are stacked between an tryptophan on one side and an leucine at the other side of the pocket. A second tryptophan shields the cap to the surrounding water.
Goette, M., Stumpe, M.C., Ficner, R. and Grubmüller, H. (2009). Molecular determinants of snurportin1 ligand affinity and structural response upon binding.
Biophys. J. 97, 581-589. [Abstract]
Strasser, A., Dickmanns, A., Lührmann, R. and Ficner, R. (2005). Structural basis for m3G-cap mediated nuclear import of spliceosomal UsnRNPs by snurportin1.
EMBO J. 24, 2235-2243. [Abstract]; PDB:[1XK5]
Strasser, A., Dickmanns, A., Schmidt, U., Penka, E., Urlaub, H., Sekine, R., Lührmann, R. and Ficner, R. (2004). Purification, crystallization and preliminary crystallographic data of the m3G cap-binding domain of human snRNP import factor snuportin1.
Acta Cryst. D60, 1628-1631. [Abstract]
3. SPN1 and binding to Importin ß
The N-terminal IBB-domain of Snurportin1 binds to the C-terminal region of Importin ß similar to the IBB domain (Fig.11.)
of a second well studied adaptor Importin alpha. Surprisingly differences in the IBB domain sequence result in differences in the dissassembly of the import complex. Whereas RanGTP binding to Importin ß bound to importin alpha requires RanGTP for release from the NPC and dissasembly, Importin ß bound to SPN1 requires RanGTP only fort the latter.
Fig.11. IBB-domain of SPN1 bound to Importin ß.
The crystal structure of the IBB-domain from SPN1 bound to Importin ß exhibits an extended confromation similar to the one observed in Fig 3 (purple structure) in contrast to the Importin ß bound to the IBB-domain of Importin alpha.
Wohlwend, D., Strasser, A., Dickmanns, A., and Ficner R. (2007). Structural Basis for RanGTP Independent Entry of Spliceosomal U snRNPs into the Nucleus.
J. Mol. Biol. 374, 1129-1138. [Abstract]; PDB:[2QNA]
4. Recycling of SPN1
Once SPN1 has released the core UsnRNPs into the nucleus, most likely within the nucleoli, it is recycled into the cytoplasm by the CRM1-dependent pathway. The binding has been shown to occur in an co-operative manner together with RanGTP.
Fig.12. CRM1 in complex with RanGTP and SPN1.
Top side view with CRM1 in green, RanGTP in Red and Snurportin1 in purple.
The complex of SPN1, RanGTP and CRM1 has been crystallized and structure analysis revealed striking differences to other karyopheris crystallized so far Fig.12.
. CRM1 forms a toroid like structure with the N- and C-terminus in close contact. Ran binds to the center of this doughnut shaped ring and interacts with both termini. Moreover SPN1, the cargo, is bound to the outer surface of CRM1 and does not interact with RanGTP, like in other structures. The structural rearrangements of CRM1 when switching from the cytoplasmic state - its structure so far hypothetical - to the nuclear state must therefore achieve structural changes at two distant sites. Besides changing from a relaxed conformation to a strained conformation causing thight interaction with RanGTP, the binding region for SPN1 has to be altered. Especially changes within the binding cleft required for binding of the first residues of SPN1 seem to be of great importance.
Fig.13. Binding of the nuclear export signature of SPN1 in the hydrophobic cleft of CRM1
The N-terminal hydrophobic residues of the SPN1 (Met1, Leu4, Leu8, Phe12, Val14) dock into a hydrophobic cleft of CRM1. Residues of SPN1 are shown as sticks (carbons in purple, oxygens in red and nitrogens in dark blue. The side chains of the hydrophobic residues pointing into the cleft are depicted as purple spheres. CRM1 is shown as a surface representation; hydrophilic areas are depicted in yellow, white denotes hydrophobic areas. The green patch cloe to Val14 marks Cys528, which is covalently modified by a CRM1-specific inhibitor, leptomycin B.
Monecke, T., Güttler, T., Neumann, P., Dickmanns, A., Görlich, D., and Ficner, R. (2009). Crystal structure of the nuclear export receptor CRM1 in complex with snurportin1 and RanGTP.
Science 324, 1087-1091. [Abstract]; PDB:[3GJX]
Import of linker histones
The nuclear import of H1 linker histones is mediated by a heterodimer of importin ß and a second receptor importin7. Interestingly, both importins may interact independantly with H1, but only as a dimer facilitate the translocation into the nucleus. The H1 binding site of importin7 comprises two extended acidic loops near the C terminus of importin7. Excperiments using isothermal titration calorimetry revealed that the formation of a receptor heterodimer in vitro is an enthalpy-driven process, whereas subsequent binding of H1 to the heterodimer is entropy-driven.
The importin ß interacting region of importin7 plays a crucial role in the activation of importin7 by importin ß. This process is allosterically regulated by importin ß and accounts for a specific tuning of the activity of the importin ß/importin7 heterodimer. The results provided new insights into cellular strategies to even energy balances in nuclear import and point toward a general regulation of importin ß-related nuclear import processes.
Wohlwend, D., Strasser, A., Dickmanns, A., Doenecke, D. and Ficner R. (2007). Thermodynamic analysis of H1 nuclear import: receptor tuning of importinbeta/importin7.
J. Biol. Chem. 282, 10707-10719. [Abstract]