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First published online 16 September 2003
doi: 10.1242/jcs.00759

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ER export of ERGIC-53 is controlled by cooperation of targeting determinants in all three of its domains

Biozentrum, University of Basel, CH-4056 Basel, Switzerland

View larger version (26K): [in a new window]   Fig. 1. A glutamine supports the ER exit determinant phenylalanine at position – 2. (A) The GM construct used for mutagenesis (Itin et al., 1995). A coiled-coil stalk domain separates the carbohydrate recognition domain (CRD) from the transmembrane domain (TMD). The di-lysine ER retrieval signal is shown in italics. Minimal anterograde transport determinants of the cytoplasmic domain are in bold (Nufer et al., 2002) (and this study). (B) Amino acid sequence of tails in GM-based constructs. Constructs 1 and 2 have been described as GMA7 and GMA5FF, respectively (Kappeler et al., 1997; Nufer et al., 2002). (C) COS cells were transfected with the indicated constructs shown in B and subjected to pulse-chase/endo H analysis using [35S]-methionine. Cells were pulsed for 10 minutes and lysed after a chase of 60 minutes. GM constructs were immunoprecipitated with anti-myc. Immunoprecipitates were treated with endo H, separated by SDS 7-10% PAGE and analyzed by fluorography. The upper band represents the endo H-resistant and the lower band the endo H-sensitive form of GM. (D) Quantification of fluorograms including that shown in C. White bar: GMA7; black bar: GMA5FF. Results are mean±s.e.m. of at least three independent experiments. * statistical significance to 2 and 4; ** statistical significance to 1 and 5 (P<0.05, Student's t-test).

View larger version (24K): [in a new window]   Fig. 2. Dimerization by disulfide bonds is required for efficient transport. (A) GM constructs. Luminal cysteines 466 and 475 of GMA7 (– tail) and GMA5FF (+ tail) (also compare Fig. 1) were changed to alanines, individually or in combination. (B) Oligomer formation of GMA7 constructs bearing cysteine substitutions. 42 hours after transfection, COS cells were labeled for 5 minutes with [35S]-methionine and chased as indicated. The cells were washed and lysed in the presence of 20 mM iodoacetamide and subjected to immunoprecipitation with anti-myc. Immunoprecipitates were separated by 4-10% gradient SDS-PAGE under nonreducing conditions followed by fluorography. Monomeric (1x), dimeric (2x) and hexameric (6x) forms of GM forms are indicated by arrows at the right margin. The size of molecular weight markers is indicated at the left margin. (C) Transport of GM constructs probed by pulse-chase/endo H (Fig. 1). Black bars: GMA7 (–) and GMA5FF (+) constructs; grey, white and hatched bars represent values of corresponding constructs with the indicated cysteine substitutions. Results are mean±s.e.m. of at least three independent experiments.

View larger version (20K): [in a new window]   Fig. 3. TMD length affects ER export. GMA7 constructs containing different TMDs were expressed in COS cells and transport was probed by pulse-chase/endo H (Fig. 1): T53, wild-type TMD of ERGIC-53; T53L2 and T53L3, T53 elongated by two or three leucines, respectively; T4, TMD of CD4; L18-L26, TMDs consisting of leucine stretches of indicated length. Results are mean±s.e.m. of at least three independent experiments. * statistical significance to all bars except L22 and T53L2; ** statistical significance to T53 (P<0.05, Student's t-test).

View larger version (31K): [in a new window]   Fig. 4. Polar and aromatic residues in the TMD are required for efficient transport. (A) Replacement of ERGIC-53's TMD slows transport. The TMD of ERGIC-53 (T53) in GM constructs with (+) or without (–) the FF motif was substituted with different amino acid repeats with a total length of 18 residues. Constructs were expressed in COS cells and transport was probed by pulse-chase/endo H (Fig. 1). Black bars: GMA7 (–) and GMA5FF (+) constructs with ERGIC-53's TMD (T53). Grey, white and hatched bars: constructs with TMD substitutions as indicated. Results are mean±s.e.m. of at least three independent experiments. (B) Representation of ERGIC-53's TMD as helical wheel. Linear sequence of TMD (residues 481-498) of human ERGIC-53 is given below the wheel. Polar amino acids and glycine 494 are in italics. Polar and aromatic residues facing one side of TMD helix are in bold. Residues influencing transport of GMA5FF are encircled in the helical wheel and marked by arrowheads in the linear sequence. (C) Transport of TMD mutants (fluorogram). COS cells were transfected with GM constructs containing either wild-type TMD (lane 1, GMA7; lane 2, GMA5FF) or amino acid substitutions in TMD of GMA5FF (lanes 3 to 6 as indicated in D). 42 hours after transfection, cells were subjected to pulse-chase/endo H analysis. (D) Quantification of fluorograms including that shown in C. Results are mean±s.e.m. of at least three independent experiments. Grey bars, GMA5FF constructs with substitutions in the TMD as indicated; white and black bars, GMA7 (wt–) and GMA5FF (wt+) with wild-type TMD. *Statistical significance to bar 2 (P<0.05, Student's t-test).

View larger version (25K): [in a new window]   Fig. 5. Substitution of polar and aromatic residues in the TMD impairs formation of disulfide-linked hexamers. (A) ERGIC-53 constructs were probed for disulfide-linked oligomer formation as described in Fig. 2 (fluorogram). The constructs contained wild-type TMD (wt TMD) or the triple substitution F484L/Q488L/Y498L (mt TMD, Fig. 4). (B) Quantification of fluorogram shown in A. Triangles, constructs with wt TMD; circles, constructs with mt TMD; filled symbols, dimers; empty symbols, hexamers. MWM, molecular weight markers. The result of a representative example is shown.

View larger version (36K): [in a new window]   Fig. 6. Reconstitution of efficient transport by a minimal number of transport determinants in a signal-less reporter. (A) GM construct and signal-less GM constructs reconstituted with a minimal set of anterograde transport determinants. The signal-less constructs possess a polyleucine TMD and a poly-alanine or poly-serine cytoplasmic domain. The minimal anterograde transport determinants are in bold. All constructs were prepared without and with alanine substitution of cysteine 466 alone, or cysteine 466 together with cysteine 475. (B) Oligomerization of reconstituted constructs. The reconstituted constructs with alanine (ala tail) or serine tail (ser tail) were tested for disulfide-linked oligomerization as described in Fig. 2, except that only one time point was analyzed (15 min chase). 1x, cysteines 466 and 475 have been substituted; 2x, cysteine 466 has been substituted; 6x, both cysteines are present. (C,D) Transport of GM constructs expressed in COS cells probed by pulse-chase/endo H (Fig. 1). rec, reconstituted constructs with cysteine substitutions indicated; sl, signal-less constructs. The `sl' and `rec' constructs contain alanine tails in panel C, and serine tails in panel D. Mean±s.e.m. of at least three independent experiments.

View larger version (62K): [in a new window]   Fig. 7. Reconstituted polar and aromatic residues mediate correct oligomerization of constructs with a poly-leucine TMD. Disulfide-linked oligomerization of different GM constructs was tested as described in Fig. 2 except that only one time point was examined (15 minutes). Monomeric (1x), dimeric (2x) and hexameric (6x) forms of GM constructs are indicated by arrows. Molecular weight markers are shown at left margin.

View larger version (42K): [in a new window]   Fig. 8. ERGIC-53 is present in a complex of high density, irrespective of the presence or absence of intermolecular disulfide bonds. (A) Separation of soluble and membrane-bound marker proteins by rate-zonal centrifugation. Shown is a representative sucrose-density gradient with the position of marker proteins in kDa. The molecular sizes of the membrane proteins CD4 (48 kDa) and sucrase-isomaltase (209 kDa) are in italics. (B) Analysis of sucrose density gradient fractions by SDS-PAGE/fluorography. Lec-1 cells stably expressing GM were pulse-labeled with [35S]-methionine for 5 minutes. After a 60 minute chase, the cells were lysed and cleared lysates were fractionated by sucrose gradient centrifugation. Fractions were immunoprecipitated with anti-myc and immunoprecipitates were separated by nonreducing SDS 4-10% PAGE followed by fluorography. Arrows indicate dimeric (2x) and hexameric (6x) forms. (C) Fractionation of GM with C466A/C475A substitution (fluorogram of a 7% SDS gel). The experiment was performed as in panel B but with Lec-1 cells stably expressing a GM variant, with C466 and C475 mutated to alanines. 1x, monomeric form.

View larger version (46K): [in a new window]   Fig. 9. Targeting determinants are conserved in ERGIC-53 orthologs. (A) Human ERGIC-53. Amino acid sequences of TMD and cytoplasmic domain are shown by a single-letter code. The di-lysine retrieval signal is in bold italic. Residues required for efficient ER exit are in bold. SS, cleavable signal sequence. (B) Partial sequence alignment of ERGIC-53 orthologs and homologs. Shown are the sequences of human ERGIC-53 (residues 453 to 510; HSapiens, SwissProt Acc P49257), and the orthologs and homologs of monkey (CAethiops, Q9TU32), rat (RNorvegicus, Q62902), mouse (MMusculus, Q9D0F3), frog (XLaevis, Q91671), fly (DMelanogaster, Q9V3A8), worm (CElegans, P90913), tunicata (PMisakiensis, Q9GR90), slime mold (DDiscoidum, Q8T2B7), baker's yeast (SCer proteins Emp47p and Emp46p, P42555 and Q12396) and fission yeast (ScPombe, O42707). The TMD is gray boxed. The conserved di-lysine ER targeting signal interacting with COPI is shown in bold italic, and the conserved ER exit motifs interacting with COPII are underlayed by black box. Residues that contribute to efficient transport are in bold, and the most membrane proximal cysteine residue (C475 in human ERGIC-53) responsible for essential dimer stabilization is framed.

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