Your friend is interested in targeting green fluorescent protein (GFP) to the ER
ID: 617194 • Letter: Y
Question
Your friend is interested in targeting green fluorescent protein (GFP) to the ER. He decides to create a modified version of GFP that he calls GFP-ER by adding the ER retention signal Lys-Asp-Glu-Leu to the C-terminus of GFP. He sees that GFP-ER is expressed in cells and that GFP-ER is four amino acids longer than the unmodified GFP. However, to his surprise, even though GFP-ER contains an ER retention signal, it is in the cytoplasm of the cell, just like the unmodified GFP. He comes to you for help in fixing this problem. What do you suggest he should do?Explanation / Answer
The endoplasmic reticulum (ER) is divided into rough and smooth domains (RER and SER). The two domains share most proteins, but RER is enriched in some membrane proteins by an unknown mechanism. We studied RER protein targeting by expressing fluorescent protein fusions to ER membrane proteins in Caenorhabditis elegans. In several cell types RER and general ER proteins colocalized, but in neurons RER proteins were concentrated in the cell body, whereas general ER proteins were also found in neurites. Surprisingly RER membrane proteins diffused rapidly within the cell body, indicating they are not localized by immobilization. Ribosomes were also concentrated in the cell body, suggesting they may be in part responsible for targeting RER membrane proteins. Go to: INTRODUCTION The endoplasmic reticulum (ER) is an extensive intracellular membrane system. It is important for a number of cellular functions including translocation of secretory proteins across the membrane, insertion of membrane proteins, lipid synthesis, calcium storage and signaling, and separation of nucleoplasm from cytoplasm. Its structure varies depending on cell type. Often two domains, rough and smooth ER (RER and SER), can be distinguished. Although this distinction has been noted for many years, nothing is known about how proteins are targeted to the two domains. In animal cells the ER forms a network that extends throughout the cell, and in several different cell types this network has been shown to be continuous. In one kind of experiment, green fluorescent protein (GFP) fused to a membrane protein that was localized in the ER, or GFP targeted to the lumen of the ER, could be bleached from the entire cell by repeatedly exposing a part of the cell to intense laser light (Cole et al., 1996 ; Subramanian and Meyer, 1997 ; Dayel et al., 1999 ). The rapidity of bleaching suggested that the proteins are freely diffusible in a continuous membrane network. In a different kind of experiment, fluorescent dye from an oil droplet diffused from directly contacted membranes into a continuous membrane network, which extended throughout both sea urchin eggs and Purkinje neurons, and is most likely the ER (Terasaki and Jaffe, 1991 ; Terasaki et al., 1994 ). In view of this continuity it is interesting to understand how domains within the ER might be established. SER and RER were initially identified by electron microscopy; the RER is decorated with ribosomes, whereas the SER is not. Although the membranes often look quite different, they were classified as domains of the same organelle because connections between the two types of membrane were observed (cf. Fawcett, 1981 ). RER must be present in all cells because in all cells nascent proteins are inserted into the membrane from ER-bound ribosomes. SER is prominent in certain cell types, such as liver, steroid-synthesizing cells, muscle, and neurons. The relationship between SER and RER composition has been best studied in liver tissue, where the two types of membranes can be separated by biochemical fractionation. Subsequent analysis of their enzyme activities and protein composition indicated that most proteins present in one domain are also found in the other (Depierre and Dallner, 1975 ; Kreibich et al., 1978 ). The major exception to the generalization that RER and SER have the same protein composition is the enrichment of several membrane proteins in the RER (Kreibich et al., 1978 ). ER membrane proteins can thus be divided between those that are concentrated in the RER, RER membrane proteins, and those that are not, general ER proteins. By fractionation of liver cells, ribophorins I and II (components of the oligosaccharyl transferase) were found to be enriched in the RER (Kreibich et al., 1978 ), as was a subunit of signal peptidase and TRAPa (SSRa; Vogel et al., 1990 ) and Sec61a (Meyer et al., 2000 ). The common feature of these proteins is that they are involved either in translocation of proteins across the ER membrane (Rapoport et al., 1996 ) or in their modification during translocation. Several studies have suggested that another membrane protein involved in the translocation process, the SRP-receptor, is not restricted to the RER (Tajima et al., 1986 ; Vogel et al., 1990 ). Thus, some, but perhaps not all, membrane proteins involved in translocation of newly synthesized proteins across the ER membrane are highly concentrated in the RER.
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