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From CellBiology

Individual assignment topic: GRASP65

What is GRASP65?

  • GRASP65 stands for Golgi reassembly stacking protein of 65kDa [1]. It has been long thought that there must exist proteins or molecules that aid the stacking of the Golgi cisternae [2][3]. GRASP65 was identified in 1993 [4]. This mammalian protein is associated with GM130, a vesicle docking protein receptor[5] [6]. It is thought to play an important role in the stacking process of the Golgi cisternae [7][8].

Location

GRASP65 is an essential stacking protein that is NEM-sensitive [9]. NEM stands for sulphydryl modifying reagent N-ethylmaleimide and is thought to interrupt the reformation of cisternae into stacks in the Golgi apparatus [10]. It has been established that GRASP65 is a N-myristoylated Golgi protein found in peripheral membrane regions of the Golgi apparatus [11]. With a combination of techniques including methods that employ antibodies as markers and immunoflourescent procedures, it was found that GRASP65 is possibly located on the cis and medial compartments of the Golgi apparatus [12][13].

GRASP65 and GM130 interaction

GRASP65 associates with GM130 to form a complex on the Golgi [14][15]. Studies have shown that this complex is very stable during cell division and interphase [16]. The GRASP65-GM130 complex contributes to the stacking process of cisternae with cooperation with p115, a docking protein [17]. When the cell divides, GRASP65 and GM130 are phosphorylated. This in turn results in the disruption between the two proteins, which disassociate allowing the unstacking process [18], which is then followed by fragmentation of the Golgi complex [19]. It is thought that the mechanism of sensitivity to sulphydryl modifying reagent N-ethylmaleimide (NEM), which hinders the reformation of Golgi fragmetns studied in a cell-free system [20]. , is most likely by steric hindrance that stops the reassembly of the GRASP65-GM150 complex and associated proteins or changes in the monomer units of the complex after cell division [21].

GRASP65 - GM130 complexes: Involvement in Golgi Dynamics

A study in 2001 by Marra et al.[22] has demonstrated that the GM130 and GRASP65 Golgi proteins are transported through a specific area in the intermediate compartment of the apparatus. Most GM120 and GRASP65 proteins are present in the center of the Golgi apparatus, but a small amount, approximately 20%, is found in the low-intermediate compartment (L-IC). The two sources of these matrix proteins interact and play a role in the morphological as well as biochemical aspects of Golgi apparatus dynamics. However, our understanding on how these proteins work and how they are regulated is still limited and requires further research.


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The diagram shows that the subdomains of the intermediate compartment are L-IC and E-IC. GM130 and GRASP65 golgi matrix proteins only exist in L-IC. Proteins secreted by the endoplasmic reticulum cross the E-IC to the L-IC either by fusion tubules with attached GRASP65-GM130 complexes extending form the Golgi complex (A) or by attachment to stations bordering L-IC (B). From the I-IC, proteins are taken up into the Golgi complex by direct fusion with the cis-Golgi cisternae (C). Another mechanism is by adjacent arrangment of the protein, following a process that flattens the protein structure, so that it can attach under the outermost cis-cisternae (D).[23]

Structure of GRASP65 & Mitosis

  • GRASP65 is a dimer[24].
  • GRASP65 protein is phosphorylated during cell division and is the major component involved out of the GRASP protein family[25]
  • The N-terminal of the GRASP domain contains amino acids 1-201. This region is vital for dimerization as well as trans-oligomerization and is not dependent on mitosis. [26]. It is highly conserved between GRASP55 and GRASP65[27], and also in different species[28].
  • The region of amino acids 202-446 contains the binding site for GM130[29].
  • The C-terminal is serine/proline-rich and contains amino acids 202-446. Although this domain does not seem to have a role in dimerization or oligomerization, it is regulated by mitotic activities, which occur because mitotic kinases phosphorylate regions on the GRASP complex.[30]
  • Towards the end of cell division with the assembling of the two identical daughter cells, GRASP65 is dephosphorylated and forms homodiamers [31].

The Future of GRASP65

There remains a lot to be studied about GRASP65 and this opens many venues for creative cell biology research. An example of possible investigation is the role of GRASP65 in the Menkes disease Gene [32]. Another interesting area of research is the involvement of the GRASP65 C-terminal in neurodegeneration[33].

References

  1. GRASP55, a second mammalian GRASP protein involved in the stacking of Golgi cisternae in a cell-free system.Shorter J, Watson R, Giannakou ME, Clarke M, Warren G, Barr FA.EMBO J. 1999 Sep 15;18(18):4949-60.PMID: 10487747 [PubMed - indexed for MEDLINE]
  2. Intercisternal material in the golgi body of Trichomonas. Amos WB, Grimstone AV. J Cell Biol. 1968 Aug;38(2):466-71. No abstract available. PMID: 5664215 [PubMed - indexed for MEDLINE]
  3. Inter- and intracisternal elements of the Golgi apparatus. A system of membrane-to-membrane cross-links.Franke WW, Kartenbeck J, Krien S, VanderWoude WJ, Scheer U, Morré DJ. Z Zellforsch Mikrosk Anat. 1972;132(3):365-80. No abstract available.PMID: 4344381 [PubMed - indexed for MEDLINE]
  4. GRASP65, a protein involved in the stacking of Golgi cisternae. Barr FA, Puype M, Vandekerckhove J, Warren G. Cell. 1997 Oct 17;91(2):253-62. PMID: 9346242 [PubMed - indexed for MEDLINE]
  5. Characterization of a cis-Golgi matrix protein, GM130. Nakamura N, Rabouille C, Watson R, Nilsson T, Hui N, Slusarewicz P, Kreis TE, Warren G. J Cell Biol. 1995 Dec;131(6 Pt 2):1715-26. PMID: 8557739 [PubMed - indexed for MEDLINE]
  6. The vesicle docking protein p115 binds GM130, a cis-Golgi matrix protein, in a mitotically regulated manner. Nakamura N, Lowe M, Levine TP, Rabouille C, Warren G. Cell. 1997 May 2;89(3):445-55. PMID: 9150144 [PubMed - indexed for MEDLINE]
  7. Characterization of a cis-Golgi matrix protein, GM130. Nakamura N, Rabouille C, Watson R, Nilsson T, Hui N, Slusarewicz P, Kreis TE, Warren G. J Cell Biol. 1995 Dec;131(6 Pt 2):1715-26. PMID: 8557739 [PubMed - indexed for MEDLINE]
  8. The vesicle docking protein p115 binds GM130, a cis-Golgi matrix protein, in a mitotically regulated manner. Nakamura N, Lowe M, Levine TP, Rabouille C, Warren G. Cell. 1997 May 2;89(3):445-55. PMID: 9150144 [PubMed - indexed for MEDLINE]
  9. Reassembly of Golgi stacks from mitotic Golgi fragments in a cell-free system. Rabouille C, Misteli T, Watson R, Warren G. J Cell Biol. 1995 May;129(3):605-18. PMID: 7730399 [PubMed - indexed for MEDLINE]
  10. Reassembly of Golgi stacks from mitotic Golgi fragments in a cell-free system. Rabouille C, Misteli T, Watson R, Warren G. J Cell Biol. 1995 May;129(3):605-18. PMID: 7730399 [PubMed - indexed for MEDLINE]
  11. GRASP55, a second mammalian GRASP protein involved in the stacking of Golgi cisternae in a cell-free system.Shorter J, Watson R, Giannakou ME, Clarke M, Warren G, Barr FA.EMBO J. 1999 Sep 15;18(18):4949-60.PMID: 10487747 [PubMed - indexed for MEDLINE]
  12. GRASP65, a protein involved in the stacking of Golgi cisternae. Barr FA, Puype M, Vandekerckhove J, Warren G. Cell. 1997 Oct 17;91(2):253-62. PMID: 9346242 [PubMed - indexed for MEDLINE]
  13. The GM130 and GRASP65 Golgi proteins cycle through and define a subdomain of the intermediate compartment.Marra P, Maffucci T, Daniele T, Tullio GD, Ikehara Y, Chan EK, Luini A, Beznoussenko G, Mironov A, De Matteis MA.Nat Cell Biol. 2001 Dec;3(12):1101-13. PMID: 11781572 [PubMed - indexed for MEDLINE
  14. GRASP65, a protein involved in the stacking of Golgi cisternae. Barr FA, Puype M, Vandekerckhove J, Warren G. Cell. 1997 Oct 17;91(2):253-62. PMID: 9346242 [PubMed - indexed for MEDLINE]
  15. The GM130 and GRASP65 Golgi proteins cycle through and define a subdomain of the intermediate compartment.Marra P, Maffucci T, Daniele T, Tullio GD, Ikehara Y, Chan EK, Luini A, Beznoussenko G, Mironov A, De Matteis MA.Nat Cell Biol. 2001 Dec;3(12):1101-13. PMID: 11781572 [PubMed - indexed for MEDLINE
  16. GRASP65, a protein involved in the stacking of Golgi cisternae. Barr FA, Puype M, Vandekerckhove J, Warren G. Cell. 1997 Oct 17;91(2):253-62. PMID: 9346242 [PubMed - indexed for MEDLINE]
  17. Characterization of a cis-Golgi matrix protein, GM130. Nakamura N, Rabouille C, Watson R, Nilsson T, Hui N, Slusarewicz P, Kreis TE, Warren G. J Cell Biol. 1995 Dec;131(6 Pt 2):1715-26. PMID: 8557739 [PubMed - indexed for MEDLINE]
  18. COP-coated vesicles are involved in the mitotic fragmentation of Golgi stacks in a cell-free system. Misteli T, Warren G. J Cell Biol. 1994 Apr;125(2):269-82. PMID: 8163545 [PubMed - indexed for MEDLINE]
  19. GRASP65, a protein involved in the stacking of Golgi cisternae. Barr FA, Puype M, Vandekerckhove J, Warren G. Cell. 1997 Oct 17;91(2):253-62. PMID: 9346242 [PubMed - indexed for MEDLINE]
  20. Characterization of a cis-Golgi matrix protein, GM130. Nakamura N, Rabouille C, Watson R, Nilsson T, Hui N, Slusarewicz P, Kreis TE, Warren G. J Cell Biol. 1995 Dec;131(6 Pt 2):1715-26. PMID: 8557739 [PubMed - indexed for MEDLINE]
  21. GRASP65, a protein involved in the stacking of Golgi cisternae. Barr FA, Puype M, Vandekerckhove J, Warren G. Cell. 1997 Oct 17;91(2):253-62. PMID: 9346242 [PubMed - indexed for MEDLINE]
  22. The GM130 and GRASP65 Golgi proteins cycle through and define a subdomain of the intermediate compartment.Marra P, Maffucci T, Daniele T, Tullio GD, Ikehara Y, Chan EK, Luini A, Beznoussenko G, Mironov A, De Matteis MA.Nat Cell Biol. 2001 Dec;3(12):1101-13. PMID: 11781572 [PubMed - indexed for MEDLINE
  23. The GM130 and GRASP65 Golgi proteins cycle through and define a subdomain of the intermediate compartment.Marra P, Maffucci T, Daniele T, Tullio GD, Ikehara Y, Chan EK, Luini A, Beznoussenko G, Mironov A, De Matteis MA.Nat Cell Biol. 2001 Dec;3(12):1101-13. PMID: 11781572 [PubMed - indexed for MEDLINE
  24. Mapping the functional domains of the Golgi stacking factor GRASP65. Wang Y, Satoh A, Warren G. J Biol Chem. 2005 Feb 11;280(6):4921-8. Epub 2004 Dec 2. PMID: 15576368 [PubMed - indexed for MEDLINE]
  25. A direct role for GRASP65 as a mitotically regulated Golgi stacking factor. Wang Y, Seemann J, Pypaert M, Shorter J, Warren G. EMBO J. 2003 Jul 1;22(13):3279-90. Erratum in: EMBO J. 2003 Aug 1;22(15):4026. PMID: 12839990 [PubMed - indexed for MEDLINE]
  26. Mapping the functional domains of the Golgi stacking factor GRASP65. Wang Y, Satoh A, Warren G. J Biol Chem. 2005 Feb 11;280(6):4921-8. Epub 2004 Dec 2. PMID: 15576368 [PubMed - indexed for MEDLINE]
  27. GRASP55, a second mammalian GRASP protein involved in the stacking of Golgi cisternae in a cell-free system. Shorter J, Watson R, Giannakou ME, Clarke M, Warren G, Barr FA. EMBO J. 1999 Sep 15;18(18):4949-60. PMID: 10487747 [PubMed - indexed for MEDLINE]
  28. Mapping the interaction between GRASP65 and GM130, components of a protein complex involved in the stacking of Golgi cisternae. Barr FA, Nakamura N, Warren G. EMBO J. 1998 Jun 15;17(12):3258-68. PMID: 9628863 [PubMed - indexed for MEDLINE]
  29. Mapping the functional domains of the Golgi stacking factor GRASP65. Wang Y, Satoh A, Warren G. J Biol Chem. 2005 Feb 11;280(6):4921-8. Epub 2004 Dec 2. PMID: 15576368 [PubMed - indexed for MEDLINE]
  30. Mapping the functional domains of the Golgi stacking factor GRASP65. Wang Y, Satoh A, Warren G. J Biol Chem. 2005 Feb 11;280(6):4921-8. Epub 2004 Dec 2. PMID: 15576368 [PubMed - indexed for MEDLINE]
  31. GRASP65, a protein involved in the stacking of Golgi cisternae. Barr FA, Puype M, Vandekerckhove J, Warren G. Cell. 1997 Oct 17;91(2):253-62. PMID: 9346242 [PubMed - indexed for MEDLINE]
  32. Pre-and Post-Transcriptional Regulation of the Menkes Disease Gene. Edward D.Harris and Sudeep Majumbar. The Journal of Trace Elements in Experimental Medicine 2003,16:181-189 DOI: 10.1002/jtra.10037
  33. A Golgi fragmentation pathway in neurodegeneration and Golgi apparatus fragmentation. Nakagomi S, Barsoum MJ, Bossy-Wetzel E, Sütterlin C, Malhotra V, Lipton SA. Neurobiol Dis. 2008 Feb;29(2):221-31. Epub 2007 Sep 7. PMID: 17964175 [PubMed - indexed for MEDLINE]

Class activities

Lecture 4 hw

  • Interesting facts about the nucleus:

The nuclear membrane is supported by a nuclear skeleton that gives the nucleus its typical round shape. chromosomes occupy certain territories that may overlap. histones condense DNA

Lecture 5 hw

  • the turnover of transport vesicles
  • visualizing SER as a meshwork of fine tubules

Lecture 7 hw

  • processes requiring energy
    • - cellular respiration
    • - cell communication
    • - gene expression/repair
    • - apoptosis

Lecture 8hw CAM = cell adhesion molecule

  • Ng-CAM: neuroglia - CAM
  • L-CAM: liver - CAM
  • I-CAM: intercellular - CAM
  • N-CAM: neuro - CAM

Lecture 10hw What is the name of the epidermal layer between the basal and granulosa layer and how does it relate to intermediate filaments? Answer: The epidermal layer is called stratum spinosum. It plays an important role in synthesising keratin to support intermediate filaments.

Lab 6 hw "If you've seen differences in the distribution of phenotypes in Tm4 over-expressing B35 cells versus control B35 cells, describe these differences. Formulate a hypothesis with regards to what changes on the molecular level may have occurred due to the over-expression of Tm4 that lead to morphological changes that you have observed"

Results:

Total for [A] Phenotypes (Tm4): A165 B10 C28 D49 E69 F8

Total for [B] Phenotypes (WT): A184 B9 C75 D51 E43 F9

Interpretation: When comparing mutant Tm4 cells with the normal B35 cells, one can conclude that they differ in cell morphology and in the structure of nutrites. This may indicate the role of Tm4 in the regulation of cell growth.

Lecture 14 hw What are the 2 main forms of generating confocal microscopy? The two main forms are laser system and spinning disk confocal. Lecture 15 hw What does "S" stand for in the S phase? S stands for "synthesis".