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Dioleoylphosphatidylcholine (DOPC) vesicles with or without 3% (mol/mol) Ptd Ins3 value of 2.6 ± 0.2 µM, which is consistent with the more robust targeting of GFP-GOLPH3 to the Golgi compared with GFP-Vps74.
GOLPH3 also binds substantially to the other tested phosphoinositides, with K-binding site.
Targeting and retention of resident integral membrane proteins of the Golgi apparatus underly the function of the Golgi in glycoprotein and glycolipid processing and sorting.
In yeast, steady-state Golgi localization of multiple mannosyltransferases requires recognition of their cytosolic domains by the peripheral Golgi membrane protein Vps74, an orthologue of human GOLPH3/GPP34/GMx33/MIDAS (mitochondrial DNA absence sensitive factor).
Alterations in this site abolish phosphoinositide binding in vitro and Vps74 function in vivo.
These results implicate Pik1 signaling in retention of Golgi-resident proteins via Vps74 and show that GOLPH3 family proteins are effectors of Golgi Ptd Ins 4-kinases.
Vps74 and GOLPH3 bind specifically to Ptd Ins4P, and a sulfate ion in a crystal structure of GOLPH3 indicates a possible phosphoinositide-binding site that is conserved in Vps74.Recruitment of Vps74 and GOLPH3 could be mediated by a direct interaction with Pik1 and/or Frq1, for which there are orthologues encoded in the human genome, or alternatively, Golgi recruitment could require the product of Pik1-Frq1, Ptd Ins4 mutant cells that express a catalytically inactive but otherwise stable form of Sac1 (Nemoto et al., 2000), GFP-Vps74 is also recruited to ER and/or PM (unpublished data), indicating that it is the loss of the phosphatase activity of Sac1 that is key to mistargeting of Vps74 and GOLPH3.