QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online June 23, 2005
doi: 10.1242/10.1242/jcs.02434

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Nair, P. Articles by Rohrer, J. Search for Related Content PubMed PubMed Citation Articles by Nair, P. Articles by Rohrer, J.

Characterization of the TGN exit signal of the human mannose 6-phosphate uncovering enzyme

Prashant Nair^1,*, Beat E. Schaub^1,*, Kai Huang², Xiang Chen², Robert F. Murphy^2,3, Janice M. Griffith⁴, Hans J. Geuze⁴ and Jack Rohrer^1,

¹ Institute of Physiology, University of Zurich, Zurich, 8057, Switzerland
² Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA
³ Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA 15213, USA
⁴ Department of Cell Biology and Institute of Biomembranes, Utrecht University School of Medicine, 3584 CX Utrecht, The Netherlands

View larger version (146K): [in a new window]   Fig. 1. Single and double immunogold labeling of UCE (B) and of UCE and CD-MPR (A,C) with gold particle sizes (in nm) as indicated in superscript on the figures. A and C show that most of the UCE and CD-MPR localize to the TGN with only a few gold particles present over cisternae of the Golgi stacks (G). In B a clathrin-coated vesicle is labeled for UCE (arrowhead) in a low-expressing cell, while in C a few coated vesicles are labeled for UCE and CD-MPR together (arrowhead). Bars, 100 nm.

View larger version (48K): [in a new window]   Fig. 2. (A) Schematic illustration of the wild-type and mutant GFP-UCE constructs. The lumenal domain of UCE was replaced with monomeric GFP fused to an N-terminal cleavable preprolactin leader sequence represented by hatched bars. The transmembrane domain of UCE is indicated by shaded bars and the amino acids in the cytoplasmic tail are shown in single letter code. Mutations of the Y488 residue and the potential exit signal residues are shown in bold letters. The position of a stop codon is indicated by an asterisk. (B) Schematic illustration of the single and combined mutant GFP-UCE constructs. The constructs have the same organization as those in Fig. 1A. In addition to the Y488 and the 502 Stop mutation, residues Q492, E493, M494 and N494 are either singly mutated or mutated simultaneously and the mutant residues are indicated by bold letters.

View larger version (36K): [in a new window]   Fig. 3. High-resolution confocal immunofluorescence images of HeLa cells expressing wild-type or mutant GFP-UCE constructs. HeLa cells were stably transfected with wild-type GFP-UCE (A), GFP-UCE 502 Stop mutant (B) or GFP-UCE Y488A/502 Stop mutant (C). The localization of the constructs was analyzed using high resolution three-dimensional confocal immunofluorescence microscopy followed by projection of z-stacks using the Imaris 4.0 software. Bars, 20 µm.

View larger version (16K): [in a new window]   Fig. 4. Dendrograms showing similarity in localization between (A) the GFP-UCE wild type, GFP-UCE 502 Stop, GFP-UCE Y488A/502 Stop and double mutant constructs of the Q492EMNGEPL sequence in the cytoplasmic tail of UCE, and (B) the wild-type and all 11 mutant constructs. The constructs are clustered based on Mahalonobis distance calculated on the SLF20 set of subcellular localization features. The similarity in pattern between any two constructs is inversely proportional to the vertical distance along the path connecting them. For example, wild-type and 502 Stop are the most similar pair of constructs. These dendrograms are available at http://murphylab.web.cmu.edu/services/PSLID/HeLa_UCE/ with a browser interface that allows the underlying images for any branch to be displayed interactively.

View larger version (146K): [in a new window]   Fig. 5. High resolution confocal immunofluorescence images of HeLa cells expressing double mutant GFP-UCE constructs of the potential TGN exit signal. HeLa cells were stably transfected with GFP-UCE Y488; Q492E-A/502 Stop (A), GFP-UCE Y488; M494N-A/502 Stop (B), GFP-UCE Y488; G496E-A/502 Stop (C) or GFP-UCE Y488; P498L-A/502 Stop (D). Bars, 20 µm.

View larger version (94K): [in a new window]   Fig. 6. High-resolution confocal immunofluorescence images of HeLa cells expressing combined and single mutant GFP-UCE constructs. HeLa cells were stably transfected with GFP-UCE Y488; Q492-A/502 Stop (A), GFP-UCE Y488; E493-A/502 Stop (B), GFP-UCE Y488; M494-A/502 Stop (C), GFP-UCE Y488; N495-A/502 Stop (D) or GFP-UCE Y488; Q492EMN-A/502 Stop mutant (E). Bars, 20 µm.