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. 2003 May 13;100(10):5783-8.
doi: 10.1073/pnas.1031748100. Epub 2003 Apr 30.

Membrane receptor trafficking: evidence of proximal and distal zones conferred by two independent endoplasmic reticulum localization signals

Sojin Shikano  1 , Min Li
Affiliations

Membrane receptor trafficking: evidence of proximal and distal zones conferred by two independent endoplasmic reticulum localization signals

Sojin Shikano et al. Proc Natl Acad Sci U S A. .

Abstract

The generic membrane trafficking signals of internal RXR and carboxyl-terminal KKXX motifs direct intracellular endoplasmic reticulum (ER) localization of the signal-bearing proteins. These signaling motifs play a critical role in partitioning proteins into designated subcellular compartments by functioning as an intracellular "zip code." In the process of determining the potential distinctions between these two otherwise functionally identical motifs, two functional zones of these signals were revealed. The KKXX signal was effective only when it was positioned closer to the membrane surface. In contrast, under identical conditions, the internal RXR signal was functional when it was positioned distally from the membrane. Different from the C-terminal KKXX signal, the internal RXR motif may be present in multiple copies. The receptor with multivalent RXR motifs displayed similar trafficking behavior to that of the same receptor with one copy of the RXR motif. The distinctive operating ranges from their anchored membrane surface provide experimental evidence for the notion that there are functional zoning layers within which membrane protein signal motifs are active.

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Figures

Figure 1
Figure 1
(A) A schematic diagram showing the coding regions of different CD4 constructs: CD4-(HA)1, CD4-(HA)3, and CD4-(HA)5. Amino acid positions are indicated at the top of the CD4-(HA)1 construct. Each of the CD4 constructs is fused with the listed protein sequences containing either the RKR motif from Kir6.2 (24) or the KKTN motif from WBP1. Specific site-directed mutations change RKR to RAA or KKTN to AATN. The number of residues that separate the transmembrane domain from the 36-aa Kir6.2 motif or WBP1 motif is indicated below each construct. (B and C) Immunoblot analyses of fusion proteins from the transfected cells. Protein lysates of transfected cells are identified by the constructs indicated at the top. The fusion protein expression was detected by anti-CD4 antibody. The molecular mass standard in kDa is indicated on the left of the gel. (D and E) FACS analyses of the surface expression are shown. The horizontal axis represents the logarithmic fluorescence intensity. The vertical axis represents the number of events (cells). Unfilled areas are signals from mock-transfected cells (negative control), and the filled areas are signals from cells transfected with the indicated CD4 constructs.
Figure 2
Figure 2
Immunocytochemistry analyses of surface expression chimeric CD4 receptors by confocal microscopy. (A) Immunostaining of 293T cells that were transiently transfected with the indicated RKR constructs (indicated on the top). Cells were stained before (Upper) and after (Lower) detergent treatments. (B) Immunostaining of 293T cells that were transiently transfected with the indicated KKTN constructs (indicated on the top). Cells were stained before (Upper) and after (Lower) detergent-mediated membrane permeation. (C) Immunostaining of COS-7 cells that were transiently transfected with the indicated RKR/RAA and KKTN/AATN constructs (indicated on the top). Cells were stained after detergent treatments.
Figure 3
Figure 3
(A) A schematic diagram of chimeric CD4 receptor with one or two copies of the Kir6.2 RKR signal. (B) FACS analyses of the surface expression are shown. The horizontal axis represents the logarithmic florescence intensity. The vertical axis represents the number of events (cells). Unfilled areas are signals from mock-transfected cells (negative control), and the filled areas are signals from cells transfected with the indicated CD4 constructs. (C) Immunoblot analyses of fusion proteins from the transfected cells. Protein lysates of transfected cells are identified by the constructs indicated at the top. The fusion protein expression was detected by anti-CD4 antibody. An overexposed blot is shown to demonstrate the absence of construct-specific degradation. The molecular mass standard in kDa is indicated on the left of the gel.
Figure 4
Figure 4
(A) A schematic diagram of the five IRK1 chimeric constructs. The site of the HA epitope insertion is as indicated. The peptide sequences fused to the C terminus are shown (Lower). (B) Different chimeric constructs were expressed and indicated (Left). The surface expression was detected by anti-HA antibody followed by FACS analyses (Left). The horizontal axis represents the logarithmic fluorescence intensity. The vertical axis represents the number of events (cells). Unfilled areas are signals from mock-transfected cells (negative control). Filled areas are the anti-HA signal. (Right) Immunofluorescence microscopy to detect the localization of indicated chimera in transfected cells by anti-HA under either permeabilized (Triton +) or nonpermeabilized (Triton −) condition.
Figure 5
Figure 5
(A) Histogram of quantification of surface expression of CD4 chimeric receptor as a function of spacing. The normalized surface expression of CD4-KKTN (Left) and RKR (Right) was determined by dividing the MESF values for RKR and KKTN constructs by those of RAA and AATN constructs (with the corresponding number of HA copies), respectively (see Materials and Methods). (B) A schematic diagram to show that the KKTN and RKR signals are active in proximal and distal zones. Repositioning the signals into a different zone by a posttranslational event including modification, folding, and protein interaction may cause the receptor to alter its subcellular localization. The membrane stop transfer signal (KR zone) immediately followed transmembrane segment is also shown.
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