Mutants of the protein serine kinase PSKH1 disassemble the Golgi apparatus.
We have dissected the molecular determinants involved in targeting the protein serine kinase PSKH1 to the endoplasmic reticulum (ER), the Golgi apparatus, and the plasma membrane (PM). Given this intracellular localization pattern, a potential role of PSKH1 in the secretory pathway was explored. The amino-terminal of PSKH1 revealed a striking similarity to the often acylated Src homology domain 4 (SH4)-harboring nonreceptor tyrosine kinases. Biochemical studies demonstrated that PSKH1 is myristoylated on glycine 2 and palmitoylated on cysteine 3. Dual amino-terminal acylation targets PSKH1 to Golgi as shown by colocalization with beta-COP and GM130, while nonpalmitoylated (myristoylated only) PSKH1 targets intracellular membranes colocalizing with protein disulphide isomerase (PDI, a marker for ER). Immunoelectron microscopy revealed that the dually acylated amino-terminal domain (in fusion with EGFP) was targeted to Golgi membranes as well as to the plasma membrane (PM), suggesting that the amino-terminal domain provides PSKH1 with membrane specificity dependent on its fatty acylation status. Subcellular fractionation by sucrose gradient analysis confirmed the impact of dual fatty acylation on endomembrane targeting, while cytosol and membrane fractioning revealed that myristoylation but not palmitoylation was required for general membrane association. A minimal region required for proper Golgi targeting of PSKH1 was identified within the first 29 amino acids. Expression of a PSKH1 mutant where the COOH-terminal kinase domain was swapped with green fluorescent protein and cysteine 3 was exchanged with serine resulted in disassembly of the Golgi apparatus as visualized by redistribution of beta-COP and GM130 to a diffuse cytoplasmic pattern, while leaving the tubulin skeleton intact. Our results suggest a structural and regulatory role of PSKH1 in maintenance of the Golgi apparatus, a key organelle within the secretory pathway.
Brede
Gaute
G
Biotechnology Centre of Oslo, University of Oslo, Gaustadalleen 21, N-0349 Oslo, Norway.
Solheim
Jorun
J
Stang
Espen
E
Prydz
Hans
H
D000215:Acylation; D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D011992:Endosomes / Q000378:metabolism / Q000648:ultrastructure; D005227:Fatty Acids; D005822:Genetic Vectors; D006056:Golgi Apparatus / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D008854:Microscopy, Electron; D009154:Mutation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D021381:Protein Transport; D014162:Transfection; D022161:Transport Vesicles / Q000378:metabolism
D005227:Fatty Acids; C417548:PSKH1 protein, human; D017346:Protein Serine-Threonine Kinases