Mobility of cytochrome P450 in the endoplasmic reticulum membrane

Abstract

Cytochrome P450 2C2 is a resident endoplasmic reticulum (ER) membrane protein that is excluded from the recycling pathway and contains redundant retention functions in its N-terminal transmembrane signal͞anchor sequence and its large, cytoplasmic domain. Unlike some ER resident proteins, cytochrome P450 2C2 does not contain any known retention͞retrieval signals. One hypothesis to explain exclusion of resident ER proteins from the transport pathway is the formation of networks by interaction with other proteins that immobilize the proteins and are incompatible with packaging into the transport vesicles. To determine the mobility of cytochrome P450 in the ER membrane, chimeric proteins of either cytochrome P450 2C2, its catalytic domain, or the cytochrome P450 2C1 N-terminal signal͞anchor sequence fused to green f luorescent protein (GFP) were expressed in transiently transfected COS1 cells. The laurate hydroxylase activities of cytochrome P450 2C2 or the catalytic domain with GFP fused to the C terminus were similar to the native enzyme. The mobilities of the proteins in the membrane were determined by recovery of f luorescence after photobleaching. Diffusion coefficients for all P450 chimeras were similar, ranging from 2.6 to 6.2 ؋ 10 ؊10 cm 2 ͞s. A coefficient only slightly larger (7.1 ؋ 10 ؊10 cm 2 ͞s) was determined for a GFP chimera that contained a C-terminal dilysine ER retention signal and entered the recycling pathway. These data indicate that exclusion of cytochrome P450 from the recycling pathway is not mediated by immobilization in large protein complexes.

Fluorescence Resonance Energy Transfer Analysis of Cytochromes P450 2C2 and 2E1 Molecular Interactions in Living Cells

“…However, we have previously established by FRAP analysis that P450 2C2/GFP has high lateral mobility in the ER membranes of transfected COS1 cells and that OEC/GFP has similar, but slightly lower, mobility (18). The aggregation of P450 2C2 observed in the present study, therefore, does not decrease its mobility, as detected by FRAP.…”

“…Although it could be argued that oligomerization of OEC in fact takes place but cannot be detected with FRET as a result of an unfavorable orientation of fluorescent tags, we consider it very unlikely. Because the enzymatic activity of OEC/GFP is very similar to that of the full-length P450 2C2/ GFP (18), both enzymes must interact functionally with P450 reductase, and in fact interaction of OEC/CFP with reductase/ YFP can be demonstrated by FRET. Thus, the heterologous transmembrane domain does not significantly affect folding of the P450s nor their membrane topology.…”

“…To make fusions of CFP and YFP to the C terminus of P450 2C1-(1-28), P450 2C1-(1-21), and P450 2E1-(1-31), BglII-HindIII inserts encoding the P450 fragments were ligated with BglII-HindIIIdigested CFP and YFP vectors. Construction of OEC/YFP and CFP was made by substituting the BamHI-NotI fragment encoding CFP or YFP from their respective vectors for the BamHI-NotI fragment encoding GFP in OEC/GFP (18). A chimera of P450 reductase/YFP was made by amplifying the P450 reductase cDNA insert from the plasmid reductase/CMV5…”

“…One mechanism that has been proposed to explain retention of ER resident proteins is that they form very large immobile complexes or networks that prevent entry of the proteins into transport vesicles. P450 2C2 is directly retained in the ER (17); however, based on fluorescence recovery after photobleaching (FRAP) studies, it has high mobility in the ER membrane, similar to that of a model protein transported from the ER (18). It is still possible that oligomerization of P450 2C2 into smaller homomeric complexes, which remain mobile in the membrane, prevents it from entering the transport vesicles.…”

“…2C2 with the GFP moiety attached to the C terminus retains its normal subcellular localization in the ER membrane (18 -20) and has catalytic activity similar to the untagged protein (18), indicating unimpaired folding in the presence of the GFP tag. Thus, fusion proteins of P450 2C2 and CFP or YFP, which are slightly modified versions of the GFP, should also be functionally similar to native P450 2C2.…”