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First published online December 11, 2006
doi: 10.1242/10.1242/jcs.03285

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Integrity of all four transmembrane domains of the tetraspanin uroplakin Ib is required for its exit from the ER

¹ Department of Cell Biology The Ronald O. Perelman Department of Dermatology and Departments of Pharmacology and Urology, NYU Cancer Institute, New York University School of Medicine, New York, NY 10016, USA
² Department of Biochemistry The Ronald O. Perelman Department of Dermatology and Departments of Pharmacology and Urology, NYU Cancer Institute, New York University School of Medicine, New York, NY 10016, USA
³ Department of Epithelial Biology Unit, The Ronald O. Perelman Department of Dermatology and Departments of Pharmacology and Urology, NYU Cancer Institute, New York University School of Medicine, New York, NY 10016, USA

View larger version (63K): [in this window] [in a new window]   Fig. 1. Construction and expression of UPIa and UPIb chimeras. (A) Sequence alignment of bovine UPIa and UPIb using Clustal W (Thompson et al., 1994). The TM domains are indicated by solid lines. The boundaries of TM domains were defined by Yu et al. (Yu et al., 1994). The conserved Glu residues in TM1 and TM3 are indicated by shading. The amino acids Tyr-Trp-Ser in the cytoplasmic C-terminal tail of UPIb are probably part of the TM4 domain and are printed in bold typeface. (B) Schematic representations of the tetraspanins UPIa and UPIb. Protein domains are numbered 1 through 9, and the amino acids as well as the numbers characterizing their positions in the polypeptide sequence that define each domain are indicated. The tetraspanins UPIa and UPIb both have four TM domains (domains 2, 4, 6 and 8), which in the tetraspanin literature are also referred to as TM1, TM2, TM3 and TM4, respectively. They have a small cytoplasmic loop (domain 5) linking TM2 and TM3 and two exoplasmic loops (domains 3 and 7). In both UPIa and UPIb, domain 7 is much larger than domain 3, and bears an N-linked oligosaccharide, as indicated by the square connected to the loop in the diagram. N- and C-termini of both tetraspanins are exposed at the cytoplasmic side of the membrane (domains 1 and 9). (C) Tables listing the abbreviations that describe the chimeric constructs generated by swapping in a systematic fashion homologous domains between UPIa and UPIb. The following rules are followed for naming the chimeras: the abbreviation of the tetraspanin (Ia for UPIa and Ib for UPIb) from which most of the domains of the chimeric protein is derived is indicated first. Replaced domains are indicated second and abbreviated by the letter a (UPIa) or b (UPIb) followed by the number assigned to the exchanged domain as defined in B. For example Ia/b7 is constructed by substituting the large loop of UPIa (domain 7) with the corresponding one from UPIb, while leaving the other domains unchanged. The amino acid number and the one letter code abbreviation characterizing the beginning and the end of the UPIa and UPIb derived domains of each construct are also listed. Asterisks indicate the chimeric proteins that could be expressed at the cell surface. (D) 293T cells expressing the UPIa/UPIb chimeras indicated in C were analyzed by western blotting using antibodies directed against the large loop of UPIa or UPIb. All chimeras are well expressed, although the ratio of the glycosylated (*) to non-glycosylated forms varied considerably.

View larger version (45K): [in this window] [in a new window]   Fig. 2. Changes in the large loop of UPIb do not interfere with exit from the ER. (A) Intact 293T cells expressing UPIa, Ib, Ib/a7, Ibm and IbCA were immunostained. In contrast to UPIa, all UPIb-related proteins are expressed on the cell surface. (B) Permeabilized 293T cells expressing UPIa, Ib, Ib/a7, Ibm and IbCA showed ER staining. (C,D) To locate the intracellular UP proteins, cells were cotransfected with the ER marker Ribophorin-I-GFP (RI-GFP) or the Golgi marker galactosyl transferase tagged with YFP (GalT-YFP) and then immunostained. The secondary antibody was conjugated to Texas Red. All the intracellularly retained UP chimeric proteins were colocalized with RI-GFP. Representative images of only UPIa and UPIb are shown here.

View larger version (55K): [in this window] [in a new window]   Fig. 3. Amino acids of the C-terminal domain of UPIb, including a crucial tyrosine residue, are tested for their function as an ER exit signal. (A) 293T cells expressing UPIb/a9, a chimeric protein in which the cytoplasmic C-terminal domain was replaced with that of UPIa. No immunostaining was observed in intact cells. (B) An alanine scan was performed on the C-terminal domain of UPIb, generating the Ala mutants UPIbA1-10. Similarly, Tyr253 was replaced with phenylalanine (UPIbF7). (C) Non-permeabilized 293T cells expressing the mutant proteins listed in B were immunostained. (D) Deletion mutants of UPIb were constructed by removing step-wise seven amino acids of the C-terminal domain 9. (E) 293T cells were transfected with UPIb or the deletion mutants indicated in D, followed by immunostaining of the non-permeabilized cells with a polyclonal anti-UPIb antibody. Only the deletion mutants that retain at least four amino acids of the C-terminal domain exit from the ER and are expressed at the cell surface. Deletion mutants Ib4 to Ib7 were retained in the ER as could be seen by immunostaining of permeabilized cells (data not shown).

View larger version (25K): [in this window] [in a new window]   Fig. 4. Kinetics of ER exit of Tac/UP chimeric proteins. (A) Schematic drawing of the Tac antigen, a type I transmembrane protein and of Tac/UP chimeric proteins. (B) HeLa cells expressing Tac/UP chimeric proteins were pulse-labeled with [35S]methionine for 15 minutes and then chased for 0 to 3 hours. The total cell lysates were harvested and subjected to immunoprecipitation using an anti-Tac antibody, followed by Endo H treatment. The Endo-H-treated samples were analyzed by SDS-PAGE and autoradiography. (C) The percentage of cells showing Endo-H resistance was calculated from densitometry values. The experiments were repeated three times to calculate s.e.m., which are indicated by the error bars. Statistical analysis was done using statistical computing software R 2.3.1.

View larger version (54K): [in this window] [in a new window]   Fig. 5. Changes in any of the four TM domains prevent UPIb from exiting from the ER. (A) Non-permeabilized 293T cells expressing UPIb, UPIa or the UPIa/UPIb chimeras indicated were analyzed by immunofluorescence microscopy. The chimeras Ib/a2, Ib/a4 and Ib/a8 were not transported to the plasma membrane, but retained in the ER (not shown). (B) Point mutants of UPIb, where the Glu32 or Glu102 were replaced by Ala (IbE32A, IbE102A), or Gln (UPIbE32Q, Ib102EQ), respectively, and Tyr99 by Ala (IbY99A), were expressed in 293T cells, followed by immunostaining of non-permeabilized cells. IbE102A was not able to reach the cell surface, indicating in addition to TM1, TM2 and TM4, the glutamic acid residue in TM3 is important for the proper assembly of the TM helices of UPIb.

View larger version (42K): [in this window] [in a new window]   Fig. 6. In contrast to UPIb, UPIa forms large aggregates in the ER, and UPIb mutants that are unable to exit from the ER sediment faster than the wild-type UPIb. Total membrane fractions from 293T cells expressing UPIa (A), UPIb (B), UPIa and UPIb (C), UPIba6 (D), UPIbE102A (E), UPIba8 (F), or UPIbA7 (G) were extracted with digitonin (1.5%) in the presence of 0.5 M NaCl. Supernatant fractions obtained after differential centrifugation were layered onto glycerol gradients (8-30%) and after centrifugation (150,000 g for 15.5 hours), aliquots of the ten gradient fractions, the sample loading zone (S) and the pellet (P) were analyzed by western blotting. Antibodies against UPIa (A,C), UPIb (B,D-F) and UPII (C) were used to localize uroplakins in the glycerol gradient. Arrowheads in A indicate the sedimentation positions of albumin (66 kDa), amylase (200 kDa) and apoferritin (443 kDa) that were used to calibrate the gradient. Asterisks indicate the peak fractions of the sedimented UP-related proteins fractions.

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