Multiple cytosolic and transmembrane determinants are required for the trafficking of SCAMP1 via an ER-Golgi-TGN-PM pathway.
How polytopic plasma membrane (PM) proteins reach their destination in plant cells remains elusive. Using transgenic tobacco BY-2 cells, we previously showed that the rice secretory carrier membrane protein 1 (SCAMP1), an integral membrane protein with four transmembrane domains (TMDs), is localized to the PM and trans-Golgi network (TGN). Here, we study the transport pathway and sorting signals of SCAMP1 by following its transient expression in tobacco BY-2 protoplasts and show that SCAMP1 reaches the PM via an endoplasmic reticulum (ER)-Golgi-TGN-PM pathway. Loss-of-function and gain-of-function analysis of various green fluorescent protein (GFP) fusions with SCAMP1 mutations further demonstrates that: (i) the cytosolic N-terminus of SCAMP1 contains an ER export signal; (ii) the transmembrane domain 2 (TMD2) and TMD3 of SCAMP1 are essential for Golgi export; (iii) SCAMP1 TMD1 is essential for TGN-to-PM targeting; (iv) the predicted topology of SCAMP1 and its various mutants remain identical as demonstrated by protease protection assay. Therefore, both the cytosolic N-terminus and TMD sequences of SCAMP1 play integral roles in mediating its transport to the PM via an ER-Golgi-TGN pathway.
School of Life Sciences, Centre for Cell and Developmental Biology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China.
Yi
Y
Cai
Tianran
T
Jia
Sheung Kwan
SK
Lam
Yu
Y
Ding
Caiji
C
Gao
Melody Wan Yan
MW
San
Peter
P
Pimpl
Liwen
L
Jiang
D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D017343:Genes, Plant; D006056:Golgi Apparatus / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D012275:Oryza / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D015398:Signal Transduction; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism; D021601:trans-Golgi Network / Q000378:metabolism
D002352:Carrier Proteins; D008565:Membrane Proteins; D010940:Plant Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins