C-terminal Dilysine Research Articles

COPI coatomer subunit α-COP interacts with the RNA binding protein Nucleolin via a C-terminal dilysine motif.

The COPI coatomer subunit α-COP has been shown to co-precipitate mRNA in multiple settings, but it was unclear whether the interaction with mRNA was direct or mediated by interaction with an adapter protein. The COPI complex often interacts with proteins via C-terminal dilysine domains. A search for candidate RNA binding proteins with C-terminal dilysine motifs yielded Nucleolin, which terminates in a KKxKxx sequence. This protein was an especially intriguing candidate as it has been identified as an interacting partner for Survival Motor Neuron protein (SMN). Loss of SMN causes the neurodegenerative disease Spinal Muscular Atrophy. We have previously shown that SMN and α-COP interact and co-migrate in axons, and that overexpression of α-COP reduced phenotypic severity in cell culture and animal models of SMA. We show here that in an mRNA independent manner, endogenous Nucleolin co-precipitates endogenous α-COP and ε-COP but not β-COP which may reflect an interaction with the so-called B-subcomplex rather a complete COPI heptamer. The ability of Nucleolin to bind to α-COP requires the presence of the C-terminal KKxKxx domain of Nucleolin. Furthermore, we have generated a point mutant in the WD40 domain of α-COP which eliminates its ability to co-precipitate Nucleolin but does not interfere with precipitation of partners mediated by non-KKxKxx motifs such as the kainate receptor subunit 2. We propose that via interaction between the C-terminal dilysine motif of Nucleolin and the WD40 domain of α-COP, Nucleolin acts an adaptor to allow α-COP to interact with a population of mRNA.

Cellular Fractionation to Characterize the Interaction of Nucleolin with Alpha-COP

Background and Hypothesis:The goal was to further characterize the interaction between Nucleolin and the alpha subunit of the COPI coatomer complex. Nucleolin contains a C-terminal dilysine motif, which mediates interactions between the WD40 domain of alpha-COP and COPI-interacting proteins. Previous work in the lab showed that this C-terminal dilysine is required for co-immunoprecipitation of alpha-COP with Nucleolin. Because alpha-COP is exclusively found in the cytoplasmic compartment, we hypothesized that the interaction between alpha-COP and Nucleolin is exclusively cytoplasmic but previous co-immunoprecipitations had only been performed from whole cell lysates. Alpha-COP has been shown to bind mRNA, but it was unclear whether this interaction was direct or whether alpha-COP was binding an RNA binding protein (RBP) which would act as a bridge between the mRNA and alpha-COP. Nucleolin acts as an RBP in both the nuclear and cytoplasmic compartments. We hypothesize that some of the mRNA bound to alpha-COP are present due to their association with Nucleolin.
 Experimental Design or Project Methods:The first aim of the project was to optimize a reproducible cell fractionation protocol to reliably separate nuclear and cytoplasmic compartments. The second goal of the project was to identify mRNA that would co-immunoprecipitated with Nucleolin in HEK293T cells where we can easily express tagged versions of alpha-COP. Nucleolin-bound mRNA had previously been identified in Hela cells. We began by testing for the expression of these mRNAs in 293-TT cells using the published RT-PCR primers.
 Results:After these tests identified Ftl (Ferritin light polypeptide) as a highly abundant transcript in 293-TTs, we performed RNA immunoprecipitation from cells expressing epitope tagged Nucleolin or alpha-COP. We confirmed that both Nucleolin and alpha-COP are in complex with Ftl mRNA.
 Potential Impact:Future experiments will use short-hairpin RNA to knockdown Nucleolin and determine whether the levels of Ftl mRNA that co-immunoprecipitated with alpha-COP are reduced in the absence of Nucleolin.

Membrane Topological Model of Glycosyltransferases of the GT-C Superfamily.

Glycosyltransferases that use polyisoprenol-linked donor substrates are categorized in the GT-C superfamily. In eukaryotes, they act in the endoplasmic reticulum (ER) lumen and are involved in N-glycosylation, glypiation, O-mannosylation, and C-mannosylation of proteins. We generated a membrane topology model of C-mannosyltransferases (DPY19 family) that concurred perfectly with the 13 transmembrane domains (TMDs) observed in oligosaccharyltransferases (STT3 family) structures. A multiple alignment of family members from diverse organisms highlighted the presence of only a few conserved amino acids between DPY19s and STT3s. Most of these residues were shown to be essential for DPY19 function and are positioned in luminal loops that showed high conservation within the DPY19 family. Multiple alignments of other eukaryotic GT-C families underlined the presence of similar conserved motifs in luminal loops, in all enzymes of the superfamily. Most GT-C enzymes are proposed to have an uneven number of TDMs with 11 (POMT, TMTC, ALG9, ALG12, PIGB, PIGV, and PIGZ) or 13 (DPY19, STT3, and ALG10) membrane-spanning helices. In contrast, PIGM, ALG3, ALG6, and ALG8 have 12 or 14 TMDs and display a C-terminal dilysine ER-retrieval motif oriented towards the cytoplasm. We propose that all members of the GT-C superfamily are evolutionary related enzymes with preserved membrane topology.

Lysine at position 329 within a C-terminal dilysine motif is crucial for the ER localization of human SLC35B4.

SLC35B4 belongs to the solute carrier 35 (SLC35) family whose best-characterized members display a nucleotide sugar transporting activity. Using an experimental model of HepG2 cells and indirect immunofluorescent staining, we verified that SLC35B4 was localized to the endoplasmic reticulum (ER). We demonstrated that dilysine motif, especially lysine at position 329, is crucial for the ER localization of this protein in human cells and therefore one should use protein C-tagging with caution. To verify the importance of the protein in glycoconjugates synthesis, we generated SLC35B4-deficient HepG2 cell line using CRISPR-Cas9 approach. Our data showed that knock-out of the SLC35B4 gene does not affect major UDP-Xyl- and UDP-GlcNAc-dependent glycosylation pathways.

α2-COP is involved in early secretory traffic in Arabidopsis and is required for plant growth.

COP (coat protein) I-coated vesicles mediate intra-Golgi transport and retrograde transport from the Golgi to the endoplasmic reticulum. These vesicles form through the action of the small GTPase ADP-ribosylation factor 1 (ARF1) and the COPI heptameric protein complex (coatomer), which consists of seven subunits (α-, β-, β'-, γ-, δ-, ε- and ζ-COP). In contrast to mammals and yeast, several isoforms for coatomer subunits, with the exception of γ and δ, have been identified in Arabidopsis. To understand the role of COPI proteins in plant biology, we have identified and characterized a loss-of-function mutant of α2-COP, an Arabidopsis α-COP isoform. The α2-cop mutant displayed defects in plant growth, including small rosettes, stems and roots and mislocalization of p24δ5, a protein of the p24 family containing a C-terminal dilysine motif involved in COPI binding. The α2-cop mutant also exhibited abnormal morphology of the Golgi apparatus. Global expression analysis of the α2-cop mutant revealed altered expression of plant cell wall-associated genes. In addition, a strong upregulation of SEC31A, which encodes a subunit of the COPII coat, was observed in the α2-cop mutant; this also occurs in a mutant of a gene upstream of COPI assembly, GNL1, which encodes an ARF-guanine nucleotide exchange factor (GEF). These findings suggest that loss of α2-COP affects the expression of secretory pathway genes.

Regulation of Ceramide Synthase by Casein Kinase 2-dependent Phosphorylation in Saccharomyces cerevisiae

Complex sphingolipids are important components of eukaryotic cell membranes and, together with their biosynthetic precursors, including sphingoid long chain bases and ceramides, have important signaling functions crucial for cell growth and survival. Ceramides are produced at the endoplasmic reticulum (ER) membrane by a multicomponent enzyme complex termed ceramide synthase (CerS). In budding yeast, this complex is composed of two catalytic subunits, Lac1 and Lag1, as well as an essential regulatory subunit, Lip1. Proper formation of ceramides by CerS has been shown previously to require the Cka2 subunit of casein kinase 2 (CK2), a ubiquitous enzyme with multiple cellular functions, but the precise mechanism involved has remained unidentified. Here we present evidence that Lac1 and Lag1 are direct targets for CK2 and that phosphorylation at conserved positions within the C-terminal cytoplasmic domain of each protein is required for optimal CerS activity. Our data suggest that phosphorylation of Lac1 and Lag1 is important for proper localization and distribution of CerS within the ER membrane and that phosphorylation of these sites is functionally linked to the COP I-dependent C-terminal dilysine ER retrieval pathway. Together, our data identify CK2 as an important regulator of sphingolipid metabolism, and additionally, because both ceramides and CK2 have been implicated in the regulation of cancer, our findings may lead to an enhanced understanding of their relationship in health and disease.

The sunflower plastidial ω3-fatty acid desaturase (HaFAD7) contains the signalling determinants required for targeting to, and retention in, the endoplasmic reticulum membrane in yeast but requires co-expressed ferredoxin for activity

Although plant plastidial omega3-desaturases are closely related to microsomal desaturases, heterologous expression in yeast of the Helianthus annuus FAD7 omega3-desaturase showed low activity in contrast to similar expression of microsomal FAD3 omega3-desaturases. However, the removal of the plastidial transit peptide and the incorporation of a KKNL motif to the C-terminus of HaFAD7 increased the activity by 10-fold compared to the native protein. N-terminal fusion of transmembrane-domains from either the yeast microsomal ELO3, (a type III signal anchor domain), or FAE1, an endoplasmic reticulum membrane anchoring domain, resulted in moderate increases in enzyme activity (5- and 7-fold, respectively), suggesting that the first, most hydrophobic transmembrane domain of HaFAD7 is sufficient to direct targeting to, and insertion into, the endoplasmic reticulum membrane. Furthermore, fusing a hemagglutinin (HA) epitope tag upstream of an endogenous C-terminal KEK motif resulted in a significant loss of activity compared to the un-tagged construct, indicating that the endogenous KEK C-terminal di-lysine motif is capable of directing in yeast the ER-retention of this normally plastidial-located protein. Western blotting analysis of constructs with internal HA epitope revealed that in whole cell extracts, with the exception of the one bound to C-terminal, it did not display a reduced level of protein accumulation. Whilst ferredoxin was shown to be required for HaFAD7 activity in yeast, it appears not necessary for protein stability and accumulation of this plastidial desaturase in the endoplasmic reticulum.

A di-arginine motif contributes to the ER localization of the type I transmembrane ER oxidoreductase TMX4

The thiol-disulfide oxidoreductases of the PDI (protein disulfide isomerase) family assist in disulfide-bond formation in the ER (endoplasmic reticulum). In the present study, we have shown that the previously uncharacterized PDI family member TMX4 (thioredoxin-like transmembrane 4) is an N-glycosylated type I membrane protein that localizes to the ER. We also demonstrate that TMX4 contains a single ER-luminal thioredoxin-like domain, which, in contrast with similar domains in other PDIs, is mainly oxidized in living cells. The TMX4 transcript displays a wide tissue distribution, and is strongly expressed in melanoma cells. Unlike many type I membrane proteins, TMX4 lacks a typical C-terminal di-lysine retrieval signal. Instead, the cytoplasmic tail has a conserved di-arginine motif of the RXR type. We show that mutation of the RQR sequence in TMX4 to KQK interferes with ER localization of the protein. Moreover, whereas the cytoplasmic region of TMX4 confers ER localization to a reporter protein, the KQK mutant of the same protein redistributes to the cell surface. Overall, features not commonly found in other PDIs characterize TMX4 and suggest unique functional properties of the protein.

Tsc10p and FVT1: topologically distinct short-chain reductases required for long-chain base synthesis in yeast and mammals

In yeast, Tsc10p catalyzes reduction of 3-ketosphinganine to dihydrosphingosine. In mammals, it has been proposed that this reaction is catalyzed by FVT1, which despite limited homology and a different predicted topology, can replace Tsc10p in yeast. Silencing of FVT1 revealed a direct correlation between FVT1 levels and reductase activity, showing that FVT1 is the principal 3-ketosphinganine reductase in mammalian cells. Localization and topology studies identified an N-terminal membrane-spanning domain in FVT1 (absent in Tsc10p) oriented to place it in the endoplasmic reticulum (ER) lumen. In contrast, protease digestion studies showed that the N terminus of Tsc10p is cytoplasmic. Fusion of the N-terminal domain of FVT1 to green fluorescent protein directed the fusion protein to the ER, demonstrating that it is sufficient for targeting. Although both proteins have two predicted transmembrane domains C-terminal to a cytoplasmic catalytic domain, neither had an identifiable lumenal loop. Nevertheless, both Tsc10p and the residual fragment of FVT1 produced by removal of the N-terminal domain with factor Xa protease behave as integral membrane proteins. In addition to their topological differences, mutation of conserved catalytic residues had different effects on the activities of the two enzymes. Thus, while FVT1 can replace Tsc10p in yeast, there are substantial differences between the two enzymes that may be important for regulation of sphingolipid biosynthesis in higher eukaryotes.

Three Homologous ArfGAPs Participate in Coat Protein I-mediated Transport

ArfGAP1 is a prototype of GTPase-activating proteins for ADP-ribosylation factors (ARFs) and has been proposed to be involved in retrograde transport from the Golgi apparatus to the endoplasmic reticulum (ER) by regulating the uncoating of coat protein I (COPI)-coated vesicles. Depletion of ArfGAP1 by RNA interference, however, causes neither a discernible phenotypic change in the COPI localization nor a change in the Golgi-to-ER retrograde transport. Therefore, we also examined ArfGAP2 and ArfGAP3, closely related homologues of ArfGAP1. Cells in which ArfGAP1, ArfGAP2, and ArfGAP3 are simultaneously knocked down show an increase in the GTP-bound ARF level. Furthermore, in these cells proteins resident in or cycling through the cis-Golgi, including ERGIC-53, beta-COP, and GM130, accumulate in the ER-Golgi intermediate compartment, and Golgi-to-ER retrograde transport is blocked. The phenotypes observed in the triple ArfGAP knockdown cells are similar to those seen in beta-COP-depleted cells. Both the triple ArfGAP- and beta-COP-depleted cells accumulate characteristic vacuolar structures that are visible under electron microscope. Furthermore, COPI is concentrated at rims of the vacuolar structures in the ArfGAP-depleted cells. On the basis of these observations, we conclude that ArfGAP1, ArfGAP2, and ArfGAP3 have overlapping roles in regulating COPI function in Golgi-to-ER retrograde transport.

Five Arabidopsis peroxin 11 homologs individually promote peroxisome elongation, duplication or aggregation

Pex11 homologs and dynamin-related proteins uniquely regulate peroxisome division (cell-cycle-dependent duplication) and proliferation (cell-cycle-independent multiplication). Arabidopsis plants possess five Pex11 homologs designated in this study as AtPex11a, -b, -c, -d and -e. Transcripts for four isoforms were found in Arabidopsis plant parts and in cells in suspension culture; by contrast, AtPex11a transcripts were found only in developing siliques. Within 2.5 hours after biolistic bombardments, myc-tagged or GFP-tagged AtPex11 a, -b, -c, -d and -e individually sorted from the cytosol directly to peroxisomes; none trafficked indirectly through the endoplasmic reticulum. Both termini of myc-tagged AtPex11 b, -c, -d and -e faced the cytosol, whereas the N- and C-termini of myc-AtPex11a faced the cytosol and matrix, respectively. In AtPex11a- or AtPex11e-transformed cells, peroxisomes doubled in number. Those peroxisomes bearing myc-AtPex11a, but not myc-AtPex11e, elongated prior to duplication. In cells transformed with AtPex11c or AtPex11d, peroxisomes elongated without subsequent fission. In AtPex11b-transformed cells, peroxisomes were aggregated and rounded. A C-terminal dilysine motif, present in AtPex11c, -d and -e, was not necessary for AtPex11d-induced peroxisome elongation. However, deletion of the motif from myc-AtPex11e led to peroxisome elongation and fission, indicating that the motif in this isoform promotes fission without elongation. In summary, all five overexpressed AtPex11 isoforms sort directly to peroxisomal membranes where they individually promote duplication (AtPex11a, -e), aggregation (AtPex11b), or elongation without fission (AtPex11c, -d).

Sorting signals in the cytosolic tail of membrane proteins involved in the interaction with plant ARF1 and coatomer.

In mammals and yeast, a cytosolic dilysine motif is critical for endoplasmic reticulum (ER) localization of type I membrane proteins. Retrograde transport of type I membrane proteins containing dilysine motifs at their cytoplasmic carboxy (C)-terminal tail involves the interaction of these motifs with the COPI coat. The C-terminal dilysine motif has also been shown to confer ER localization to type I membrane proteins in plant cells. Using in vitro binding assays, we have analyzed sorting motifs in the cytosolic tail of membrane proteins, which may be involved in the interaction with components of the COPI coat in plant cells. We show that a dilysine motif in the -3,-4 position (relative to the cytosolic C-terminus) recruits in a very specific manner all the subunits of the plant coatomer complex. Lysines cannot be replaced by arginines or histidines to bind plant coatomer. A diphenylalanine motif in the -7,-8 position, which by itself has a low ability to bind plant coatomer, shows a clear cooperativity with the dilysine motif. Both dilysine and diphenylalanine motifs are present in the cytosolic tail of several proteins of the p24 family of putative cargo receptors, which has several members in plant cells. The cytosolic tail of a plant p24 protein is shown to recruit not only coatomer but also ADP ribosylation factor 1 (ARF1), a process which depends on both dilysine and diphenylalanine motifs. ARF1 binding increases twofold upon treatment with brefeldin A (BFA) and is completely abolished upon treatment with GTPgammaS, suggesting that ARF1 can only interact with the cytosolic tail of p24 proteins in its GDP-bound form.

Differential oligomerization of endoplasmic reticulum-retained connexin43/connexin32 chimeras.

To examine early events in connexin oligomerization, we made connexin constructs containing a C-terminal di-lysine based endoplasmic reticulum (ER) retention/retrieval signal (HKKSL). Previously, we found that both Cx32-HKKSL and Cx43-HKKSL were retained in the ER. However, Cx32-HKKSL oligomerized into hexameric hemichannels, but Cx43-HKKSL was retained as an apparent monomer. To define elements that prevent Cx43-HKKSL oligomerization in the ER, we made a series of HKKSL-tagged Cx43/Cx32 chimeras. When expressed by HeLa cells, some chimeras were retained in the ER as apparent monomers, whereas others oligomerized in the ER. To date, the second and third transmembrane domains and the cytoplasmic loop domain provide the minimal sufficient Cx43 element to inhibit ER oligomerization.

Recent advances in the study of tomato Cf resistance genes

Recent advances in the study of tomato <i>Cf</i> resistance genes

Endoplasmic reticulum membrane-sorting protein of lymphocytes (BAP31) is highly expressed in neurons and discrete endocrine cells.

BAP31 is a transmembrane protein that associates with nascent membrane proteins in transit between endoplasmic reticulum (ER) and cis-Golgi. Its C-terminal dilysine (KKEE) motif, mediating return to the ER, is consistent with a role in early sorting of membrane proteins. An initiator caspase-binding site in the C-terminal domain of BAP31 is implicated in cytoplasmic membrane fragmentation events of apoptosis. Although BAP31 RNA is ubiquitous, the protein's anatomic localization has not been determined. To gain further insight into its possible functions, we localized BAP31 in primate tissues using monoclonal antibodies. Immunoreactivity was prominent in T- and B-lymphocytes in blood and in thymus, in cerebellar Purkinje neuron bodies and dendrites, in gonadotrophs of the anterior pituitary, ovarian thecal and follicular cells, active but not quiescent thyroid epithelium, adrenal cortex more than medulla, and proximal more than distal renal tubules. Blood vessels and skeletal muscle were nonreactive. The anatomic distribution of BAP31 and the nature of proteins identified thus far as its cargo exiting the ER, suggest an interaction with proteins assembling in macromolecular complexes en route to selected sites of exocytotic and signaling activities. Apoptotic associations in mature tissues could be physiological (lymphocytes, endocrine cells) or pathological (Purkinje neurons, renal tubules).

The C-terminal dilysine motif for targeting to the endoplasmic reticulum is not required for Cf-9 function.

The tomato resistance gene Cf-9 encodes a membrane-anchored, receptor-like protein that mediates specific recognition of the extracellular elicitor protein AVR9 of Cladosporium fulvum. The C-terminal dilysine motif (KKRY) of Cf-9 suggests that the protein resides in the endoplasmic reticulum. Previously, two conflicting reports on the subcellular location of Cf-9 were published. Here we show that the AARY mutant version of Cf-9 is still functional in mediating AVR9 recognition, suggesting that functional Cf-9 resides in the plasma membrane. The data presented here and in reports by others can be explained by masking the dilysine signal of Cf-9 with other proteins.

Maturation and Endosomal Targeting of β-Site Amyloid Precursor Protein-cleaving Enzyme: THE ALZHEIMER'S DISEASE β-SECRETASE

The amyloidogenic Abeta peptide is liberated from the amyloid precursor protein (APP) by two proteolytic activities, beta-secretase and gamma-secretase. Recently, a type I membrane protein termed BACE (beta-site APP cleaving enzyme) with characteristics of an aspartyl protease has been identified as the beta-secretase. We undertook a series of biochemical and morphological investigations designed to characterize the basic properties of this protein. Initial studies indicated that BACE undergoes N-linked glycosylation at three of four potential sites. Metabolic pulse-chase experiments revealed that after core glycosylation, BACE is rapidly and efficiently transported to the Golgi apparatus and distal secretory pathway. BACE was also found to be quite stable, being turned over with a t(12) of approximately 16 h. Retention of BACE in the endoplasmic reticulum by introduction of a C-terminal dilysine motif prevented complex carbohydrate processing and demonstrated that propeptide cleavage occurs after exit from this organelle. BACE exhibited intramolecular disulfide bonding but did not form oligomeric structures by standard SDS-polyacrylamide gel electrophoresis analysis and sedimented as a monomer in sucrose velocity gradients. Immunofluorescence studies showed a largely vesicular staining pattern for BACE that colocalized well with endosomal, but not lysosomal, markers. Measurable levels of BACE were also detected on the plasma membrane by both immunostaining and cell surface biotinylation, and cycling of the protein between the cell membrane and the endosomes was documented. A cytoplasmic dileucine motif was found to be necessary for normal targeting of BACE to the endosomal system and accumulation of the protein in this intracellular site.

The C-terminal dilysine motif confers endoplasmic reticulum localization to type I membrane proteins in plants.

The tomato Cf-9 disease resistance gene encodes a type I membrane protein carrying a cytosolic dilysine motif. In mammals and yeast, this motif promotes the retrieval of type I membrane proteins from the Golgi apparatus to the endoplasmic reticulum (ER). To test whether the C-terminal KKXX signal of Cf-9 is functional as a retrieval motif and to investigate its role in plants, green fluorescent protein (GFP) was fused to the transmembrane domain of Cf-9 and expressed in yeast, Arabidopsis, and tobacco cells. The fusion protein was targeted to the ER in each of these expression systems, and mutation of the KKXX motif to NNXX led to secretion of the fusion protein. In yeast, the mutant protein reached the vacuole, but plants secreted it as a soluble protein after proteolytic removal of the transmembrane domain. Triple hemagglutinin (HA)-tagged full-length Cf-9 was also targeted to the ER in tobacco cells, and cleavage was also observed for the NNXX mutant protein, suggesting an endoprotease recognition site located within the Cf-9 lumenal sequence common to both the GFP- and the HA-tagged fusions. Our results indicate that the KKXX motif confers ER localization in plants as well as mammals and yeast and that Cf-9 is a resident protein of the ER.

The C-Terminal Dilysine Motif Confers Endoplasmic Reticulum Localization to Type I Membrane Proteins in Plants

The C-Terminal Dilysine Motif Confers Endoplasmic Reticulum Localization to Type I Membrane Proteins in Plants

Mobility of cytochrome P450 in the endoplasmic reticulum membrane.

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 signal/anchor sequence and its large, cytoplasmic domain. Unlike some ER resident proteins, cytochrome P450 2C2 does not contain any known retention/retrieval 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 signal/anchor sequence fused to green fluorescent 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 fluorescence after photobleaching. Diffusion coefficients for all P450 chimeras were similar, ranging from 2.6 to 6.2 x 10(-10) cm2/s. A coefficient only slightly larger (7.1 x 10(-10) cm2/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.