3209709

From CellBiology

p115 GOLGIN PROTEIN

Structure

p115 was originally discovered in the year of 1992 using the Rothman intra-Goligi transport assay.[1] p115 is derived from a Golgin proteins family. Often Golgins functioning as thether proteins in membrane fusion and providing structural supports for Golgi apparatus. Thether proteins are long fibrous proteins with extreme coiled-coil domains.[2][3] p115 is one of the best characterized mammalian thethering proteins. It is a stable myosin-shaped homodimer with a large globular N-terminal head region (N-terminus), a coiled-coil tail central region, and a short carboxyl-terminal acidic region (C-terminus).[3] It is a type of peripheral membrane proteins and is localized in ER-Golgi Intermediate Compartments (ERGICs), cis-Golgi cisternae and cis-Golgi network (CGN).[1][4] p115 is also known as transcytosis-associated proteins.[5]

Functions

p115 act as a vesicle thethering factor in intra-Golgi transport.[1][3][6] and ER-to-Golgi transport.[4][7] In addition, p115 is involved in regrowth or staking of Golgi fragments during postmitotic formation.[8] To enable its function, p115 co-operates with other golgins such as GM130 and giantin. On the other hand, p115 acts to control the structural organization of Golgi apparatus.[3][6].[4]


Vesicle thethering and fusion at the cis-Golgi

Firstly, Golgi reassembly and stacking proteins (GRASP65s) which are newly synthesized bind to a C-terminus of GM130 and assemble into a complex in the cytoplasm. The GRASP65/GM130 complex then directly target to cis-Golgi membrane.[9][10] Rab1-related GTPase (Rab1- related guanosine triphosphatase) recruited p115 by binding to the central region of p115 and is loaded on the incoming vesicle. p115 interacts with N-terminal 74 amino acids of GM130 [11][12] and N-terminal 70 amino acids of giantin </ref>[12] to dock and thether ER-derived COPI-coated vesicles to the cis-Golgi membrane. [13] Thus, a giantin/p115/GM130 thethering complex is formed. The thethering complex precedes to direct the cognate COPI vesicle to assemble with its target soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNAREs) complex necessary for membrane fusion.[14] Therefore, the p115 functions in retrograde recycling COPI-coated vesicles via the giantin.[13]

Alternatively, p115 is recruited onto ER-derived COPII-coated vesicles with the help of Rab1 to promote the clustering of COPII-coated vesicles.[15] Vesicle tubular clusters (VTCs) formed.[15] The activity of p115 at the VTCs stage is independent of any GM130 or giantin.[7] The acidic C-terminus of p115 subsequently binds to basic N-terminus of GM130,[11] dock the VTCs to the cis-Golgi membrane.[15] p115 targeting the COPII-coated vesicles bind to SNAREs, aid in the formation of cis-SNARE complex. Therefore, the p115 promotes anterograde transport from ER to Golgi apparatus.[16]

Nevertheless, p115 mediated clustering of COPII-coated vesicles, docking of COPI-coated vesicles and COPII-coated vesicles, thethering of cisternae, and SNARE pin assembly.[17] See Figure 1.

Vesicle tethering at the cis-Golgi membrane


Post-mitotic Golgi reassembly

p115 is dependent of GM130 for Golgi fragment reassembly after the mitotic fragmentation of Golgi apparatus.[8] p115 binds to Golgi apparatus only when GM130 is dephosphorylated not phosphorylated and thus Golgi fragments reassemble.[18] To permit regrowth and stacking of Golgi cisternae, p115 need to be added together N-ethylmaleimide–sensitive fusion protein (NSF), α-and γ-soluble NSF attachment proteins (SNAPs).[19]


Structural integrity of Golgi apparatus

p115 plays a key role in maintaining the structural integrity of Golgi apparatus.[3][6].[4] Mammalian Golgi apparatus undergoes extensive irreversible fragmentation during apoptosis and eventually cell death. [20] During apoptosis, p115 was selectively cleaved by initiator caspases 3 and executioner caspases 8 to generate 205 residues of C-terminus fragment (CTF).[21] p115 CTF expressed is translocated into nucleus, act as a pro-apoptotic effect to induce early apoptosis program.[21] p115 is not completely cleaved until the later times of apoptosis activity, yet no Golgi fragmentation observed.[21] Efficiency of p115 cleavage is then enhanced by SUMolytion and therefore, increased the kinetics of Golgi fragmentation.[22]

Depletion of p115

Inhibit VSV-G protein transport

As known, p115, GM130 and giantin are involved in the ER-to-Golgi traffic. In vesicular stomatitis virus temperature sensitive 045 (VSV-G ts 045) based semi-intact cell assay, cell depleted of p115 inhibited the VSV-G protein transport from ER to Golgi. [7] The transport is inhibited before mannosidase II processing.[23] N-terminal 74 amino acids of GM130 and N-terminal 70 amino acids of giantin were the potent transport inhibitors.[23] Both compete with each other for p115 binding on the Golgi complex.[12] However, both of them do not function simultaneously.[23] Furthermore, a mutant of GM130 lacking the corresponding p115 binding site lead to binding failure of p115.[11] The mutant GM130 expression inhibited VSV-G transport to the plasma membrane significantly.[9] As a result, Golgi cisternae disappear and number of Golgi vesicles accumulated in Golgi area increased.[6] Consequently, inhibition of anterograde ER-to-Golgi protein transport highlighted the importance of p115.[7]

Diassembly of Golgi cisternae

Besides that, phosphorylation of GM130 during mitosis inhibited p115 to bind to Golgi membrane. [11] At the prophase, metaphase and anaphase of mitosis, GM130 is phosphorylated and thus Golgi fragments disassemble.[24][25][26] Conversely, at telophase, GM130 is dephosphorylated and subsequently Golgi fragments start to reassemble.[27] Cdc2 kinase acts in control the state of GM130 on serine 25. [28] Removal of the p115 binding site on the Golgi membrane causes Golgi fragments disassemble and so prevent thethering and docking of COPI-coated vesicles.[11][18][24] As a result, transport through the Golgi apparatus is inhibited during mitosis.[18] Cisternae are converted into vesicles by continuous budding of vesicles without fussion.[29] Consumption of the cisternal membrane is correlated with increased number of Golgi vesicles.[6] Contrastly, VSV-G protein transport from ER-to-Golgi still occurred under mitotic conditions.[6]

Current Research

To determine, CTF acts as a co-factor to modify expression of apoptotic genes. Shaeri Mukherjee and Dennis Shields (2009) Nuclear Import Is Required for the Pro-apoptotic Function of the Golgi Protein p115. J. Biol. Chem., 284(3): 1709-1717. [1]

REFERENCES

  1. 1.0 1.1 1.2 Waters, M.G., Clary, D.O., and Rothman, J.E. (1992) A novel 115-kD peripheral membrane protein is required for intercisternal transport in the Golgi stack. J. Cell Biol. 118, 1015–1026.>
  2. Nakajima, H., Hirata, A., Ogawa, Y., Yonehara, T., Yoda, K., and Yamasaki, M. (1991) J. Cell Biol. 113,245 -260.>
  3. 3.0 3.1 3.2 3.3 3.4 Sapperstein, S. K., Lupashin, V. V., Schmitt, H. D., and Waters, M. G. (1996) Assembly of the ER to Golgi SNARE complex requires Uso1pJ. Cell Bio., 132: 755 -767.>
  4. 4.0 4.1 4.2 4.3 Nelson, D.S., Alvarez, C., Gao, Y.-s., García-Mata, R., Fialkowski, E., and Sztul, E. (1998) The membrane transport factor TAP/p115 cycles between the Golgi and earlier secretory compartments and contains distinct domains required for its localization and function. J. Cell Biol., 143: 319–331.>
  5. Barroso M., Nelson, D.S., and Sztul, E. (1995) Transcytosis-associated protein (TAP)/p115 is a general fusion factor required for binding of vesicles to acceptor membranes. Proc. Natl Acad. Sci. USA, 92: 527–531.>
  6. 6.0 6.1 6.2 6.3 6.4 6.5 Joachim Seemann, Eija Jamsa Jokitalo, and Graham Warren (2000) The Role of the Tethering Proteins p115 and GM130 in Transport through the Golgi Apparatus In Vivo. Mol Biol Cell., 11(2): 635–645.>
  7. 7.0 7.1 7.2 7.3 Barros Alvarez, C., Fujita, H., Hubbard, A., and Sztul, E (1999) ER to Golgi transport: Requirement for p115 at a pre‐Golgi VTC stage. J. Cell Biol. 147: 1205–1222.>
  8. 8.0 8.1 Shorter, J., and Warren, G (1999) A role for the vesicle tethering protein, p115, in the postmitotic stacking of reassembling Golgi cisternae in a cell‐free system. J. Cell Biol. 146: 57–70.>
  9. 9.0 9.1 F.A. Barr, N. Nakamura and G. Warren (1998) Mapping the interaction between GRASP65 and GM130, components of a protein complex involved in the stacking of Golgi cisternae. EMBO J., 17: 3258–3268.>
  10. S.-I. Yoshimura, N. Nakamura, F.A. Barr, Y. Misumi, Y. Ikehara, H. Ohno, M. Sakaguchi and K. Mihara (2001) Direct targeting of cis-Golgi matrix proteins to the Golgi apparatus. J Cell Sci, 114: 4105–4115.>
  11. 11.0 11.1 11.2 11.3 11.4 Nakamura N, Lowe M, Levine TP, Rabouille C, Warren G. (1997) The vesicle docking protein p115 binds GM130, a cis-Golgi matrix protein, in a mitotically regulated manner. Cell, 89: 445–455.>
  12. 12.0 12.1 12.2 Linstedt, A. D., Jesch, S. A., Mehta, A., Lee, T. H., Garcia-Mata, R., Nelson, D. S., and Sztul, E. (2000) J. Biol. Chem,. 275: 10196-10201.>
  13. 13.0 13.1 Sonnichsen, M. Lowe, T.P. Levine, E. Jamsa, B. Dirac-Svestrup and G. Warren (1998) A role for giantin in docking COPI vesicles to Golgi membranes. J Cell Biol., 140: 1013–1021.>
  14. Shorter, M.B. Beard, J. Seeman, A.B. Dirac-Svestrup and G. Warren (2002) Sequential tethering of golgins and catalysis of SNAREpin assembly by the vesicle-tethering protein p115. J Cell Biol 157: 45–62.>
  15. 15.0 15.1 15.2 B.B. Allan, B.D. Moyer and W.E. Balch (2000) Rab1 recruitment into a cis-SNARE complex: programming budding COPII vesicles for fusion. Science 289: 444–448.>
  16. Beard M, Satoh A, Shorter J, Warren G. (2005) A cryptic Rab1-binding site in the p115 tethering protein. J Biol Chem., 280:25840–25848.>
  17. Merran C. Derby and Paul A. Gleeson (2007). New Insights into Membrane Trafficking and Protein Sorting. International Review of Cytology, 261: 47 0074-7696/07.>
  18. 18.0 18.1 18.2 Lowe M, Nakamura N, Warren G. (1998a) Golgi division and membrane traffic. Trends Cell Biol., 8: 40–44.>
  19. C. Rabouille, T.P. Levine, J.-M. Peters and G. Warren (1995) An NSF-like ATPase, p97, and NSF mediate cisternal regrowth from mitotic Golgi fragments. Cell, 82: 905–914.>
  20. Maag, R. S., Hicks, S. W., and Machamer, C. E. (2003) Curr. Opin. Cell Biol. 15, 456-461.>
  21. 21.0 21.1 21.2 Raymond Chiu, Leonid Novikov, Shaeri Mukherjee, and Dennis Shields (2002) A caspase cleavage fragment of p115 induces fragmentation of the Golgi apparatus and apoptosis. J Cell Biol., 159(4): 637–648.>
  22. Shaeri Mukherjee and Dennis Shields (2009) Nuclear Import Is Required for the Pro-apoptotic Function of the Golgi Protein p115. J. Biol. Chem., 284(3): 1709-1717.>
  23. 23.0 23.1 23.2 Alvarez, C.I., Garcia-Mata, R., Hauri, H.P., and Sztul, E.S. (2001) The p115-interactive proteins, GM130 and giantin participate in ER-Golgi traffic. J. Biol. Chem. 276: 2693-2700>
  24. 24.0 24.1 Lowe M, Rabouille C, Nakamura N, Watson R, Jackman M, Jamsa E, Rahman D, Pappin DJ, Warren G (1998b) Cdc2 kinase directly phosphorylates the cis-Golgi matrix protein GM130 and is required for Golgi fragmentation in mitosis. Cell, 94: 783–793.> Cite error: Invalid <ref> tag; name "L1_24" defined multiple times with different content
  25. T.P. Levine, C. Rabouille, R.H. Kieckbusch and G. Warren (1996) Binding of the vesicle docking protein p115 to Golgi membranes is inhibited under mitotic conditions. J. Biol. Chem., 271: 17304–17311.>
  26. Shima DT, Haldar K, Pepperkok R, Watson R, Warren G. (1997) Partitioning of the Golgi apparatus during mitosis in living HeLa cells. J Cell Biol, 137: 1211–1228.>
  27. Lucocq J.M., Berger E.G., Warren G (1989) Mitotic Golgi fragments in HeLa cells and their role in the reassembly pathway. J. Cell Biol., 109: 463–474.>
  28. Lowe M., Rabouille C., Nakamura N., Watson R., Jackman M., Jamsa E., Rahman D., Pappin D.J., Warren G (1998) Cdc2 kinase directly phosphorylates the cis-Golgi matrix protein GM130 and is required for Golgi fragmentation in mitosis. Cell, 94: 783–793.>
  29. Cabrera-Poch N, Pepperkok R, Shima DT (1998) Inheritance of the mammalian Golgi apparatus during the cell cycle. Biochim Biophys Acta., 1404: 139–151.>

LECTURE FEEDBACK

Nucleus

What did you find interesting and did not know about the nucleus?

It was so amazing to know of how cell nucleus assembled. It can be just a little cell or organelle but scientist actually able to recognized each of its compartment or component in a nucleus cell. The nuclear cell identified from nuclear envelope which consist of outer and inner nuclear membrane, then nucleolus, chromosome territories, interchromatin, speckles interchromatin, and finally nuclear bodies. DIfferent components play a different role which may be essential or non essential in a cell. Cajal bodies is so amazing to learn because of its size of 0.1 microns to 2.0 microns only.

Exocytosis

What concept about exocytosis did you find difficult to understand?

The concept of how a protein or lipid move from one organelle to other organelle, especially the golgi apparatus which is my own topic for my project. I found it quite hard to understand with the limited information about the Golgi apparatus that involved in exocytosis.

Mitochondria

What types of cellular processes require lots of energy from the mitochondria?

Cell movement, cell signalling, cell metabolism, and cell respiration.

Adhesion

What do the different "CAM" acronyms stand for?

I-CAM - Intercellular Cell Adhesion Molecule

L-CAM - Liver Cell Adhesion Molecule

N-CAM - Neural Cell Adhesion Molecule

Ng-CAM - Neural Glial Cell Adhesion Molecule

Intermediate Filaments

What is the name of the epidermal layer between the basal and granulosa layer and how does it relate to intermediate filaments?

The epidermal layer is called stratum spinosum and it function to synthesis cytokeratin to support the intermediate filaments.

Lab 6 - Cystokeleton

"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"

Total for [A] Phenotypes: 173 A - 11 B - 32 C - 51 D - 54 E - 17 F - 8

Total for [B] Phenotypes: 192 A - 5 B - 90 C - 42 D - 38 E - 13 F - 5

Since genotype "A" and genotype "B" will be compared and analysed for altered formation of processes.

Genotype A depicted lower neurites in the B35 cells compared to the genotype B. This showed the roles of Tm4 in regulating specific cell structures

Confocal Microscopy

What are the 2 main forms of generating confocal microscopy?

Laser system and spinning disk confocal.


Cell Cycle

What does "S" stand for in the S phase?

It is refer to synthesis in the S phase.