Targeting of OSBP-related protein 3 (ORP3) to endoplasmic reticulum and plasma membrane is controlled by multiple determinants.
The intracellular targeting determinants of oxysterol binding protein (OSBP)-related protein 3 (ORP3) were studied using a series of truncated and point mutated constructs. The pleckstrin homology (PH) domain of ORP3 binds the phosphoinositide-3-kinase (PI3K) products, PI(3,4)P2 and PI(3,4,5)P3. A functional PH domain and flanking sequences are crucial for the plasma membrane (PM) targeting of ORP3. The endoplasmic reticulum (ER) targeting of ORP3 is regulated the by a FFAT motif (EFFDAxE), which mediates interaction with VAMP-associated protein (VAP)-A. The targeting function of the FFAT motif dominates over that of the PH domain. In addition, the exon 10/11 region modulates interaction of ORP3 with the ER and the nuclear membrane. Analysis of a chimeric ORP3:OSBP protein suggests that ligand binding by the C-terminal domain of OSBP induces allosteric changes that activate the N-terminal targeting modules of ORP3. Notably, over-expression of ORP3 together with VAP-A induces stacked ER membrane structures also known as organized smooth ER (OSER). Moreover, lipid starvation promotes formation of dilated peripheral ER (DPER) structures dependent on the ORP3 protein. Based on the present data, we introduce a model for the inter-relationships of the functional domains of ORP3 in the membrane targeting of the protein.
Lehto
Markku
M
Department of Molecular Medicine, National Public Health Institute, Biomedicum, P.O. Box 104, FI-00251 Helsinki, Finland.
Hynynen
Riikka
R
Karjalainen
Katja
K
Kuismanen
Esa
E
Hyvärinen
Kati
K
Olkkonen
Vesa M
VM
D020816:Amino Acid Motifs; D002352:Carrier Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000378:metabolism*; D050556:Fatty Acid-Binding Proteins; D006801:Humans; D050356:Lipid Metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000378:metabolism; D017354:Point Mutation; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism*; D000098825:Oxysterol Binding Proteins
D002352:Carrier Proteins; D050556:Fatty Acid-Binding Proteins; D008565:Membrane Proteins; D010716:Phosphatidylinositols; D033921:Vesicular Transport Proteins; C471934:OSBPL3 protein, human; D000098825:Oxysterol Binding Proteins; C114051:VAPA protein, human; C118303:phosphoinositide-3,4,5-triphosphate; C118301:phosphoinositide-3,4-bisphosphate