Chapter 14. Examining KDEL in a GFP Construct

Introduction

Analyze the Data
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Analyze the Data 14-2: Examining KDEL in a GFP Construct

You have genetically engineered a green fluorescent protein (GFP) construct containing a KDEL sequence. When the construct is transfected into normal human fibroblasts and examined using fluorescence microscopy, the fluorescence appears throughout the cytoplasm, as diagrammed below.

Question

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It’s everywhere because GFP is not a secreted protein so it wouldn’t have a hydrophobic signal sequence to get it into the RER in the first place. The KDEL sequence would not be recognized because the protein itself is not in the right compartment.

Question

b. To analyze the results further, fractions of different organelles and the cytoplasm are collected from cells expressing this KDEL-containing GFP construct and then examined on Western blots using antibodies against GFP (27 kDa) and protein disulfide isomerase (PDI), a resident rough ER (RER) protein of approximately 55 kDa.

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This would be expected for any resident ER protein; some of it might be lost as vesicles move to the Golgi. Luckily, these proteins have a KDEL sequence that is recognized by the KDEL receptor and the protein is shuttled back to the RER. Since PDI is an essential protein involved in catalyzing disulfide bonds between proteins, many proteins not having this modification would not fold properly and would therefore have abnormal or no function. If both alleles of a PDI gene were knocked out, the mouse would not survive, as PDI is an essential enzyme.

Question

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Any antibody to a specific nuclear protein (e.g., histones).

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