Vivo में और अडैप्टर क्लैथ्रिन इंटरेक्शन की इन विट्रो अध्ययन में
Summary
क्लैथ्रिन की मध्यस्थता endocytosis अनुकूलक प्रोटीन है कि माल चयन और क्लैथ्रिन कोट विधानसभा समन्वय पर निर्भर करता है. यहाँ हम प्रक्रियाओं का वर्णन करने के लिए एडाप्टर क्लैथ्रिन शारीरिक संपर्क और जीना सेल इमेजिंग दृष्टिकोण एक मॉडल के रूप में खमीर endocytic अनुकूलक प्रोटीन Sla1p का उपयोग का अध्ययन.
Abstract
A major endocytic pathway initiates with the formation of clathrin-coated vesicles (CCVs) that transport cargo from the cell surface to endosomes1-6. CCVs are distinguished by a polyhedral lattice of clathrin that coats the vesicle membrane and serves as a mechanical scaffold. Clathrin coats are assembled during vesicle formation from individual clathrin triskelia , the soluble form of clathrin composed of three heavy and three light chain subunits7,8. Because the triskelion does not have the ability to bind to the membrane directly, clathrin-binding adaptors are critical to link the forming clathrin lattice to the membrane through association with lipids and/or membrane proteins9. Adaptors also package transmembrane protein cargo, such as receptors, and can interact with each other and with other components of the CCV formation machinery9.
Over twenty clathrin adaptors have been described, several are involved in clathrin mediated endocytosis and others localize to the trans Golgi network or endosomes9. With the exception of HIP1R (yeast Sla2p), all known clathrin adaptors bind to the N-terminal -propeller domain of the clathrin heavy chain9. Clathrin adaptors are modular proteins consisting of folded domains connected by unstructured flexible linkers. Within these linker regions, short binding motifs mediate interactions with the clathrin N-terminal domain or other components of the vesicle formation machinery9. Two distinct clathrin-binding motifs have been defined: the clathrin-box and the W-box9. The consensus clathrin-box sequence was originally defined as L[L/I][D/E/N][L/F][D/E]10 but variants have been subsequently discovered11. The W-box conforms to the sequence PWxxW (where x is any residue).
Sla1p (Synthetic Lethal with Actin binding protein-1) was originally identified as an actin associated protein and is necessary for normal actin cytoskeleton structure and dynamics at endocytic sites in yeast cells12. Sla1p also binds the NPFxD endocytic sorting signal and is critical for endocytosis of cargo bearing the NPFxD signal13,14. More recently, Sla1p was demonstrated to bind clathrin through a motif similar to the clathrin box, LLDLQ, termed a variant clathrin-box (vCB), and to function as an endocytic clathrin adaptor15. In addition, Sla1p has become a widely used marker for the endocytic coat in live cell fluorescence microscopy studies16. Here we use Sla1p as a model to describe approaches for adaptor-clathrin interaction studies. We focus on live cell fluorescence microscopy, GST-pull down, and co-immunoprecipitation methods.
Protocol
Discussion
क्लैथ्रिन लेपित vesicles (CCV) endocytosis और पार Golgi नेटवर्क से परिवहन में भाग लेने और endosomes, संरक्षित मार्ग है कि यूकेरियोटिक कोशिका जीव विज्ञान के लिए मौलिक हैं. इस तरीके कागज में वर्णित दृष्टिकोण CCV गठन के लिए जिम्म…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
लोमो डॉक्टरेट फैलोशिप के लिए एक NSF पुल द्वारा समर्थित है. इस काम में इस्तेमाल सूक्ष्मदर्शी भाग में माइक्रोस्कोप इमेजिंग नेटवर्क कोलोराडो राज्य विश्वविद्यालय से कोर बुनियादी ढांचे अनुदान द्वारा समर्थित है. लेखक की प्रयोगशाला में क्लैथ्रिन एडेप्टर पर काम शुरू हुआ धन और अमेरिकन हार्ट एसोसिएशन एसडी 09SDG2280525 पुरस्कार CSU द्वारा समर्थित है
Materials
| Material | Company |
|---|---|
| QuickChange-XL site-directed mutagenesis kit | Stratagene |
| protease inhibitor cocktail | Sigma |
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