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The production of post-Golgi vesicles requires a protein kinase C-like molecule, but not its phosphorylating activity
| Content Provider | Semantic Scholar |
|---|---|
| Author | Simon, Jan Philipp Ivanov, Ivan E. Adesnik, Milton Sabatini, David D. |
| Copyright Year | 1996 |
| Abstract | We have recently described a system that recreates in vitro the generation of post-Golgi vesicles from purified Golgi fractions obtained from virus-infected MDCK cells in which the vesicular stomatitis virus-G envelope glycoprotein had been allowed to accumulate in vivo in the TGN. Vesicle formation, monitored by the release of the viral glycoprotein, was shown to require the activation of a GTP-binding ADP ribosylation factor (ARF) protein that promotes the assembly of a vesicle coat in the TGN, and to be regulated by a Golgi-associated protein kinase C (PKC)-like activity. We have now been able to dissect the process of post-Golgi vesicle generation into two sequential stages, one of coat assembly and bud formation, and another of vesicle scission, neither of which requires an ATP supply. The first stage can occur at 20 degrees C, and includes the GTP-dependent activation of the ARF protein, which can be effected by the nonhydrolyzable nucleotide analogue GTP gamma S, whereas the second stage is nucleotide independent and can only occur at a higher temperature of incubation. Cytosolic proteins are required for the vesicle scission step and they cannot be replaced by palmitoyl CoA, which is known to promote, by itself, scission of the coatomer-coated vesicles that mediate intra-Golgi transport. We have found that PKC inhibitors prevented vesicle generation, even when this was sustained by GTP gamma S and ATP levels reduced far below the K(m) of PKC. The inhibitors suppressed vesicle scission without preventing coat assembly, yet to exert their effect, they had to be added before coat assembly took place. This indicates that a target of the putative PKC is activated during the bud assembly stage of vesicle formation, but only acts during the phase of vesicle release. The behavior of the PKC target during vesicle formation resembles that of phospholipase D (PLD), a Golgi-associated enzyme that has been shown to be activated by PKC, even in the absence of the latter's phosphorylating activity. We therefore propose that during coat assembly, PKC activates a PLD that, during the incubation at 37 degrees C, promotes vesicle scission by remodeling the phospholipid bilayer and severing connections between the vesicles and the donor membrane. |
| Starting Page | 355 |
| Ending Page | 370 |
| Page Count | 16 |
| File Format | PDF HTM / HTML |
| PubMed reference number | 8896594 |
| Volume Number | 135 |
| Journal | The Journal of cell biology |
| Alternate Webpage(s) | http://ftp.ncbi.nlm.nih.gov/pub/pmc/ae/78/jc1352355.PMC2121038.pdf |
| Alternate Webpage(s) | http://jcb.rupress.org/content/jcb/135/2/355.full.pdf |
| Language | English |
| Access Restriction | Open |
| Subject Keyword | ADP-Ribosylation Factors Adenosine Diphosphate Adenosine Triphosphate Analog Bud - CHV concept COPI-Coated Vesicles Coated vesicle Golgi Apparatus Guanosine Triphosphate Lindane Murine sarcoma viruses Nucleotides Occur (action) Phospholipase D Phospholipids Protein Kinase C Protein Kinases Stage Is Stage level 1 Stage level 2 Staphylococcal Protein A TG gene Tissue membrane Vesicle (morphologic abnormality) Vesicular Stomatitis Virus Diseases membrane budding physical hard work retrograde vesicle-mediated transport, Golgi to ER vesicle coat |
| Content Type | Text |
| Resource Type | Article |
