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Intracellular trafficking of MAN1, an integral protein of the nuclear envelope inner membrane

Wei Wu, Feng Lin and Howard J. Worman*

Departments of Medicine and of Anatomy and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA



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  		Fig. 1. Cellular localization of full-length MAN1 and CHL. (A) Proteins expressed by the plasmid constructs FL and FG-CHL, which were used to transfect COS-7 cells. Domains of proteins are represented as follows: yellow, FLAG-tag; white, nucleoplasmic, luminal and C-terminal domains of MAN1; red, LEM domain; orange, the first transmembrane segment of MAN1; dark green, the second transmembrane segment of MAN1; pink, transmembrane segment of CHL; light blue, luminal domain of CHL. The N-terminus of each protein is at the left. (B) Nuclear rim fluorescence characteristic of inner nuclear membrane labeling is seen for full-length MAN1 (FL) and a labeling pattern characteristic of outer nuclear membrane, ER and endosomes is seen for CHL (FG-CHL). Cells were fixed and immunostained with anti-FLAG monoclonal antibodies which were recognized by FITC-conjugated secondary antibodies. Bar, 10 µm.

 


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  		Fig. 2. The N-terminal domain of MAN1 contains a nuclear envelope targeting signal. (A) Proteins expressed by the plasmid constructs MAN538, MAN476-CHL and CT, which were used to transfect COS-7 cells. Domains of proteins are represented by: yellow, FLAG-tag; white, nucleoplasmic, luminal and C-terminal domains of MAN1; red, LEM domain; orange, the first transmembrane segment of MAN1; dark green, the second transmembrane segment of MAN1; pink, transmembrane segment of CHL; light blue, luminal domain of CHL. The N-terminus of each protein is on the left. (B) Cellular localizations of the N-terminal domain with the first transmembrane segment of MAN1 (MAN538), the N-terminal domain of MAN1 (amino acids 1-476) fused to truncated CHL (MAN476-CHL) and the two transmembrane segments and nucleoplasmic, C-terminal domain of MAN1 (CT) as determined by laser scanning confocal immunofluorescence microscopy. Antibodies used were anti-FLAG monoclonal antibodies recognized by FITC-conjugated secondary antibodies (green, left panels labeled FLAG) and polyclonal antibodies against lamin B1 recognized by rhodamine-conjugated secondary antibodies (red, middle panels labeled Lamin B1). The panels on the right show an overlay of the FITC and rhodamine channels, with areas of overlap appearing yellow. Bar, 10 µm.

 


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  		Fig. 3. The nucleoplasmic, N-terminal domain of MAN1 plus its first transmembrane domain is localized to the inner nuclear membrane using digitonin-permeabilized cells. (A) Protein expressed by the plasmid construct MAN538-GFP, which was used to transfect COS-7 cells. Domains of the protein are represented by: yellow, FLAG-tag; white, nucleoplasmic, luminal and C-terminal domains of MAN1; red, LEM domain; orange, the first transmembrane segment of MAN1; green, GFP. The N-terminus of the protein is on the left. (B) Fluorescence microscopic analysis of COS-7 cells that were transfected with plasmid expressing the FLAG-tagged N-terminal domain and first transmembrane segment of MAN1 fused to GFP. The cells were permeabilized with Triton X-100 (upper panels, labeled Triton X-100) or ice-cold digitonin (lower panels, labeled Digitonin) and were then labeled with anti-FLAG monoclonal antibody. The left panels (GFP) show fluorescence from GFP (green), the middle panels (FLAG) show signal from anti-FLAG monoclonal antibodies recognized by rhodamine conjugated secondary antibodies (red) and the right panels (Overlay) show an overlay of the GFP and FLAG panels, with areas of overlap appearing yellow. Bar, 10 µm.

 


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  		Fig. 4. The entire N-terminal domain of MAN1 is necessary for efficient inner nuclear membrane targeting and retention. (A) Proteins expressed by the plasmid constructs MAN{Delta}8-199-CHL, MAN{Delta}85-336-CHL and MAN351-CHL, which were used to transfect COS-7 cells. Domains of the proteins are represented by: yellow, FLAG-tag; white, nucleoplasmic domain of MAN1; red, LEM domain; pink, transmembrane segment of CHL; light blue, luminal domain of CHL. Thin lines indicate portions of MAN1 deleted from each protein. The N-terminus of each protein is on the left.

(B) Immunofluorescence images from laser scanning confocal immunofluorescence microscopy showing localization of MAN1-CHL chimeric proteins from which amino acids 8-199 (MAN{Delta}8-199-CHL), 85-336 (MAN{Delta}85-336-CHL) and 351-476 (MAN351-CHL) of MAN1 were deleted. Antibodies used were anti-FLAG monoclonal antibodies recognized by FITC-conjugated secondary antibodies (green, left panels labeled FLAG) and polyclonal antibodies against lamin B1 recognized by rhodamine-conjugated secondary antibodies (red, middle panels labeled Lamin B1). The panels to the right show an overlay of the FITC and rhodamine channels, which areas of overlap appearing yellow. Bar, 10 µm.

 


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  		Fig. 5. Size of the nucleocytoplasmic domain of MAN1 affects its inner nuclear membrane targeting. (A) Protein expressed by the plasmid CMPK-MAN538, which was used to transfect COS-7 cells. Domains of the protein are represented by: yellow, FLAG-tag; dark blue, truncated CMPK; white, nucleoplasmic domain of MAN1; red, LEM domain; orange, the first hydrophobic transmembrane segment of MAN1. The N-terminus of the protein is on the left. (B) Immunofluorescence images from laser scanning confocal immunofluorescence microscopy showing localization of truncated CMPK fused to the N-terminal domain and first transmembrane segment of MAN1. Antibodies were anti-FLAG monoclonal antibodies recognized by FITC-conjugated secondary antibodies (green, upper and lower left panels labeled FLAG), polyclonal antibodies against lamin B1 (labeled Lamin B1) or against SSR-alpha (labeled SSR), which is a marker for ER protein, recognized by rhodamine-conjugated secondary antibodies (red, middle panels). The right panels show an overlay of the FITC and rhodamine channels, with areas of overlap appearing yellow. Bar, 10 µm.

 


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  		Fig. 6. The lateral diffusional mobility of MAN1 is decreased in the nuclear envelope compared with the ER. Confocal fluorescence studies of localization and mobilities of the MAN1-GFP fusion protein encoded by plasmid MAN538-GFP (see legend to Fig. 3) measured by qualitative FRAP, showing photobleach recovery in (A) nuclear envelope membranes and in (B) ER membranes. The fluorescence in the boxed regions was bleached and the fluorescence recovery is shown at 10, 60 and 290 seconds after the bleaching. Bar, 5 µm.

 


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  		Fig. 7. Comparison of FRAP experiments for the MAN1-GFP fusion protein (see Fig. 6) in the ER and nuclear envelope membranes. Fluorescence intensities in recovery after photobleaching are plotted versus time for the fusion protein in the ER (x) and nuclear envelope (o). Data points were obtained at 2 second intervals. The curves displayed kinetics allowing for the determination of diffusion constants for MAN1-GFP in the ER and nuclear envelope. Fluorescence intensity was normalized to prebleach intensity and corrected for total loss of fluorescence because of the high-energy laser bleach to I0=100 (normalized prebleach intensity).

 




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