3224277

From CellBiology

Nup 358/RanBP2

Structure

Nup 358/RanBP2 is a large 358 kD molecular weight protein that is a major constituent of the nucleopore complex (NPC)[1]. It has been demonstrated experimentally that it resides on the cytoplasmic side of the NPC.[2] Delphin et al (1997) report the structural form of purified Nup 358/Ran BP2 as being rod-like and flexible. They also postulate that it is either: a major contributor, or even the entire contribution of the eight filaments projecting into the cytoplasm from the periphery of the NPC[3].

Located along this filament - like nucleoporin are a number of varied functional domains. These include: 4 Ran binding domains [4] zinc finger motifs.[5],a domain with FG (phenylalanine and glycine) repeats (typical of most discovered nucleoporins), a leucine rich region, a cyclophilin domain and a kinesin binding domain.[6]. It is thought that at its base, Nup 358/Ran BP2 is anchored to the nucleopore complex via an interaction with the nuceloporins Nup 214 and Nup 88[7]. As aformentioned the structure of RanBP2 is such that it has many functional domains, therefore it is unsurprisingly involved in many cellular processes. At present some of the functions have been elucidated, but much of the functional domain activity of this structurally complex protein remains unknown.

Function

Nup 358/Ran BP2 is a multi-functional protein. It performs different functions depending on which stage of progression the cell cycle is in. In interphase it is primarily involved in nuclear import and export and the sumoylation of protein cargoes. In mitosis RanBP2 carries out its other functions of kinetochore recruitment and possible mitochondrial transport and function effects.


Interphase functioning

  • Nuclear import/export

Review the 'Nuclear transport' sub-section on the ANAT3231 Cell Biology 2009 Group 9 project page([1])

In summary:

Ran BP2 is an acronym for Ran Binding Protein 2[2]. The name itself provides an insight into what RanBP2 does. Therefore, it is important to understand what Ran is. Ran is a small GTP-binding protein that binds to karyopherins; it undertakes an essential role in the majority of transport pathways between the nucleus and cytoplasm. RanGTP is synthesized in the nucleus as a product of RCC1 (a chromatin bound exchange factor) RanGAP 1 in conjunction with the RBP1 (Ran Binding Protein 1) protein or the homologous RANBP1 domain of RanBP2 facilitate the breakdown of GTP on Ran.In general, this ability of RanBP2 to faciliatate the hydrolysis of GTP on Ran implicates it in import and export processes as the majority of these processes are driven by the Ran GTPase cycle.[8]

Moreover, In nuclear import pathways regulated by importin alpha/beta it has been shown in vivo that without sufficient levels of RanBP2, nuclear import is greatly compromised. [9]
RanBP2 is integral to transport pathways regulated by importin beta (Hutten, S., Flotho, A., Melchior, F. and Kehlenbach, R. H.(2008). The Nup358-RanGAP complex is required for efficient importin{alpha}/{beta}-dependent nuclear import. Molecular Biology of the Cell 19, 2300-2310).

Similarly, Ran BP2 is also integral to protein export. It was aforementioned that RanBP2 exhibits a zinc finger domain. It has been shown experimentally that this domain is a specific docking site for exportin-1 a protein that is integral to exportin mediated nuclear export. [10]


  • Sumoylation of proteins

Sumoylation is a process, which alters the functionality of a protein. This process is similar to ubiquination in that they are both examples of post-translational modification (modification occurring after initial protein translation). However, in sumoylation, SUMO (small ubiquitin-like modifier) proteins are added to protein instead of ubiquitin units.[11] RanBP2 is described as being an E3 SUMO1 ligase. This is due to its ability to interact with the E2 protein Ubc9, together they modify the protein SP100 via the addition of SUMO 1. SUMO 1 is a protein which has a variety of functions. But its association with RanBP2 is such that it targets RanGAP1 to RanBP2/Nup358. This is quite significant as RanBP2 will only interact with RANGAP1 that has been sumoylated.[12]

Mitotic functions

Nuclear envelope breakdown

It has been documented that RanBP2/Nup 358 in conjunction with another nucleoporin Nup 153 (localised at the nuclear basket) function in the breaking down of the the nuclear envelope in mitosis. [13].

Kinetochore recruitment

It has also been observed that RanBP2 performs some function associated with the kinetochore in mitosis. Experiments have shown there to be high levels of RanBP2 found on kinetochores, at this stage of the cell cycle. RanBP2 forms a complex with RanGAP1 and together they function to stabilise microtubule attachments and regulate the addition of other entities such as Mad1 and Mad 2 to the kinetochore.[14].

Mitochondrial interaction

Another recent study has also shown that RanBP2 may have some kind of role in mitochondrial transport and function. Its exact association and the mechanism is as yet unknown. However it has been shown that deficiency of RanBP2 can have a number of detrimental effects such as: the downregulation of glucose catabolism, impairment of energy maintainance and the delocalising of mitochondrial constituents of photosensory neurons, it is thought that this maybe dude to a loss of the functioning of the kinesin binding domain of RanBP2/Nup 358.[15]Hence, implicating RanBP2 in correct mitochondrial functioning.

Table: Summary of RanBP2 Functions

Abnormalities associated with RanBP2/Nup 358

There are many abnormalities associated with deficient levels or improper functioning of RanBP2, these include:

  • Arresting of mitosis if RanBP2 is not present. [16]
  • A missense mutation in the gene that encodes for Ran Binding Protein 2, has been observed to result in acute necrotizing encephalopathy (ANE). [17]
  • RanBP2 has been shown to be an integral component to the proper functioning of photoreceptor cells. Insufficiency of RanBP2 has been linked to cancer, aneuploidy and degradation of these cells. [18]

Future

Even though the available literature documents many of the currently known functions of RanBP2. There is one central notion that keeps emerging. That is, that much of the functioning of this essential protein is still unknown, this is largely due to the multiple functional domains that it possess.

One particularly exciting prospect of future research in RanBP2, is investigating its possible tumor suppressing qualities. [19]


Glossary

NPC - Nucleopore complex

Nucleoporin - Protein structure which is a constituent of the nucleopore complex.

RAN GAP1 - A protein which activates the GTPase activity of Ran

Sumoylation - Process by which SUMO (Small Ubiquitin-like Molecules) are added to a protein to alter its activity

SUMO 1 - Is a ubiquitin like protein that is added to proteins in the case of RanBP2; it must be added to RanGap1 for RanBP2 to interact with it.

RANBP1 - Ran-binding protein 1

GTP - Guanine tri-phosphate an energy rich molecule that when hydrolysed produces energy, it is a component of the Ran cycle the powers nucleocytoplasmic transport.

RanGTP - Name given to complex of Ran protein bound to GTP molecule.

E3 - Is the third stage in the sumoylation cascade.

Aneuploidy - An abnormal number of chromosomes

References

  1. Yokoyama, N., Hayashi, N., Seki, T., Pante, N., Ohba, T., Nishii, K., Kuma, K., Hayashida, T., Miyata, T., Aebi, U., Fukui, M., and Nishimoto, T. (1995). A giant nucleopore protein that binds Ran/TC4. Nature 376, 184 –188.PMID:7603572
  2. Walther, T. C., Pickersgill, H. S., Cordes, V. C., Goldberg, M. W.,Allen, T. D., Mattaj, I. W. and Fornerod, M.(2002). The cytoplasmic filaments of the nuclear pore complex are dispensable for selective nuclear protein import. Journal of Cell Biology. 158, 63-77.PMID 12105182
  3. Delphin C, Guan T, Melchior F, Gerace L. RanGTP targets p97 to RanBP2, a filamentous protein localized at the cytoplasmic periphery of the nuclear pore complex. Molecular Biology of the Cell. 1997 December; 8(12):2379–2390.PMID 9398662
  4. Wu, J., Matunis, M.J., Kraemer, D., Blobel, G., and Coutavas, E. (1995). Nup358, a cytoplasmically exposed nucleoporin with pep-tide repeats, Ran-GTP binding sites,zinc finger motifs, a cyclophilin A domain, a kinesin binding domain and a leucine-rich region. Journal Biol. Chem. 270, 14209 –14213.PMID 7775481
  5. Villa Braslavsky CI, Nowak C, Görlich D, Wittinghofer A, Kuhlmann J., (2000).Different structural and kinetic requirements for the interaction of Ran with the Ran-binding domains from RanBP2 and importin-beta. Biochemistry .39, 11629-11639.PMID 10995230
  6. Wu, J., Matunis, M.J., Kraemer, D., Blobel, G., and Coutavas, E. (1995). Nup358, a cytoplasmically exposed nucleoporin with pep-tide repeats, Ran-GTP binding sites, zinc fingers, a cyclophilin A homologous domain, and a leucine-rich region. Journal Biol. Chem. 270, 14209 –14213.PMID 7775481
  7. Bernad, R., van der Velde, H., Fornerod, M., and Pickersgill, H. (2004). Nup358/RanBP2 attaches to the nuclear pore complex via association with Nup88 and Nup214/CAN and plays a supporting role in CRM1-mediated nuclear protein export. Molecular Cell Biology 24, 2373–2384.PMID 14993277
  8. Geiss-Friedlander, R. & Melchior, F. Concepts in sumoylation: a decade on. Nature Reviews Molecualr Cell Biology. 8, 947–956 (2007).PMID 18000527
  9. Hutten, S., Flotho, A., Melchior, F. and Kehlenbach, R. H.(2008). The Nup358-RanGAP complex is required for efficient importin{alpha}/{beta}-dependent nuclear import. Molecular Biology of the Cell 19, 2300-2310.PMID 18305100
  10. The zinc finger cluster domain of RanBP2 is a specific docking site for the nuclear export factor, exportin-1. Singh, B.B., Patel, H.H., Roepman, R., Schick, D., Ferreira, P.A. J. Biol. Chem. (1999)PMID 10601307
  11. Geiss-Friedlander, R. & Melchior, F. Concepts in sumoylation: a decade on. Nature Reviews Molecualar Cell Biology. 8, 947–956 (2007).PMID 18000527
  12. Mahajan, R., Delphin, C., Guan, T., Gerace, L., and Melchior, F. (1997). A small ubiquitin-related polypeptide involved in targeting RanGAP1 to nuclear pore complex protein RanBP2. Cell 88, 97–107. 965–971. PMID 9019411
  13. A.J. Prunuske, J. Liu, S. Elgort, J. Joseph, M. Dasso and K.S. Ullman, Nuclear envelope breakdown is coordinated by both Nup358/RanBP2 and Nup153, two nucleoporins with zinc finger modules, Mol. Biol. Cell 17 (2006), pp. 760–769.PMID 16314393
  14. J. Joseph, S.T. Liu, S.A. Jablonski, T.J. Yen, M. Dasso, The RanGAP1–RanBP2 complex is essential for microtubule–kinetochore interactions in vivo, Curr. Biol. 14 (2004) 611–617.PMID: 15062103
  15. Cho KI, Cai Y, Yi H, Yeh A, Aslanukov A, Ferreira PA., (2007).Association of the kinesin-binding domain of RanBP2 to KIF5B and KIF5C determines mitochondria localization and function.. Traffic. 8, 1722-35.PMID: 17887960
  16. Salina D, Enarson P, Rattner JB, Burke B., (2003).Nup358 integrates nuclear envelope breakdown with kinetochore assembly.. Journal of cell biology. 162, 991-1000. PMID 12963708
  17. Neilson DE, Adams MD, Orr CM, Schelling DK, Eiben RM, Kerr DS, Anderson J, Bassuk AG, Bye AM, Childs AM, Clarke A, Crow YJ, Di Rocco M, Dohna-Schwake C, Dueckers G, Fasano AE, Gika AD, Gionnis D, Gorman MP, Grattan-Smith PJ, Hackenberg A, Kuster A, Lentschig MG, Lopez-Laso E, Marco EJ, Mastroyianni S, Perrier J, Schmitt-Mechelke T, Servidei S, Skardoutsou A, Uldall P, van der Knaap MS, Goglin KC, Tefft DL, Aubin C, de Jager P, Hafler D, Warman ML., (2009).Infection-triggered familial or recurrent cases of acute necrotizing encephalopathy caused by mutations in a component of the nuclear pore, RANBP2.. American Journal of Human Genetics 84, 44-51.
  18. Cho KI, Yi H, Yeh A, Tserentsoodol N, Cuadrado L, Searle K, Hao Y, Ferreira PA., (2008).Haploinsufficiency of RanBP2 is neuroprotective against light-elicited and age-dependent degeneration of photoreceptor neurons.. Cell death and differentiation. 16, 287-97.PMID 18949001
  19. Navarro MS, Bachant J., (2008).RanBP2: a tumor suppressor with a new twist on TopoII, SUMO, and centromeres. Cancer Cell. 2008 Apr;13(4):293-5.

Homework Items

1. Lecture 4 - Nucleus - What did you find interesting and did not know about the nucleus? In my previous study of the nucleus in biology.

I had never previously encountered cajal bodies or PML bodies. The PML bodies particularly interest me as their function is as yet unknown.

2. Lecture 5 - Exocytosis - What concept about exocytosis did you find difficult to understand? I found it difficult to understand what it is that allows the transport vesicles to move?

3. Lecture 7 - Mitochondria - What types of cellular processes require lots of energy from the mitochondria?

  • Apoptosis
  • Muscle contraction
  • Glycolysis
  • Cellular respiration

4. Lecture 8 - Adhesion - What do the different "CAM" acronyms stand for? CAM is an acronym for Cell Adhesion Molecules, there are different types of CAMs including:

  • N-CAM = Neural cell adhesion molecule.
  • Ng-CAM = Neural glial cell adhesion molecule.
  • L-CAM = Liver cell adhesion molecule.
  • I-CAM = Intercellular adhesion molecule.


5. Lecture 10 - What is the name of the epidermal layer between the basal and granulosa layer and how does it relate to intermediate filaments?

The name of the epidermal layer between the basal and granulosa epidermal layers is the stratum spinosum. The stratum spinsosum layer relates to intermediate filaments, through its production of cytokeratins. Which are intermediate filaments that are composed of keratin.



6. Lecture 14 - Confocal Microscopy' - What are the 2 main forms of generating confocal microscopy?

The two main forms of confocal microscopy are spinning disk and laser scanning: The spinning disk technique: utilises a spinning disk with multiple pinholes to achieve confocality. This enables rapid image capture. The laser technique is the more conventional technique. It only uses one aperture to achieve confocality. The advantage of the laser is that it achieve higher image quality.


7. Lecture 15 - Cell Cycle - What does "S" stand for in the S phase? The S stands for 'Synthesis'.

  1. Lab 10 - Peer Review - Individual Projects