From Cytosol to the Apoplast: The Hygromycin Phosphotransferase (HYG(R)) Model in Arabidopsis.
The process by which proteins are secreted via endoplasmic reticulum (ER)/Golgi-independent mechanism is conveniently called unconventional protein secretion. Recent studies have revealed that unconventional protein secretion operates in plants, but little is known about its underlying mechanism and function. This chapter provides methods we have used to analyze unconventional character of hygromycin phosphotransferase (HYG(R)) secretion in plant cells. Following isolation of protoplasts from HYG (R) -GFP-transgenic plants and incubation with brefeldin A (BFA), an inhibitor of conventional secretory pathway, we easily obtain protein extracts from protoplasts and culture medium separately. These proteins are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by Western blot analysis with anti-GFP antibodies.
Key Laboratory of Plant Resources, Institute of Botany, Chinese Academy of Sciences, Beijing, 100093, China. hyz@ibcas.ac.cn.
Haiyan
H
Zhang
Key Laboratory of Plant Resources, Institute of Botany, Chinese Academy of Sciences, Beijing, 100093, China.
Jinjin
J
Li
D017360:Arabidopsis / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D020126:Brefeldin A / Q000494:pharmacology; D002458:Cell Fractionation; D003600:Cytosol / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000378:metabolism*; D021381:Protein Transport; D011523:Protoplasts / Q000378:metabolism*; D055571:Secretory Pathway
D010940:Plant Proteins; D020126:Brefeldin A; D017853:Phosphotransferases (Alcohol Group Acceptor); C063242:hygromycin-B kinase
GFP antibodies; Hygromycin phosphotransferase; Protoplast isolation; Trichloroacetic acid precipitation; Unconventional protein secretion; Western blot