Limits...
Induced oligomerization targets Golgi proteins for degradation in lysosomes.

Tewari R, Bachert C, Linstedt AD - Mol. Biol. Cell (2015)

Bottom Line: These were targeted to the cis- and trans-Golgi, respectively, using domains from mannosidase-1 and galactosyltransferase.Significantly, upon oligomerization, each redistributed to peripheral punctae and was degraded.This occurred in the absence of detectable UPR activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213.

No MeSH data available.


Related in: MedlinePlus

Induced oligomerization causes degradation of galactosyltransferase. (A) Images show thresholded GFP fluorescence of GT-FM and anti-giantin antibody staining after the indicated times of AP washout. Bar, 5 μm. (B) Quantified number of cytoplasmic punctae per cell is shown for GT-FM and the giantin control at the indicated time points after AP washout (mean ± SEM, n = 3, >10 cells/experiment). (C) Total cell fluorescence levels of GT-FM and giantin were determined using ImageJ and normalized to the level of AP treated cells before washout (mean ± SEM, n = 3, >10 cells/experiment). (D, E) Blot and quantification showing level of GT-FM in cell extracts prepared at the indicated times after AP washout with GPP130 as a loading control (mean ± SEM, n = 3).
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4666137&req=5

Figure 9: Induced oligomerization causes degradation of galactosyltransferase. (A) Images show thresholded GFP fluorescence of GT-FM and anti-giantin antibody staining after the indicated times of AP washout. Bar, 5 μm. (B) Quantified number of cytoplasmic punctae per cell is shown for GT-FM and the giantin control at the indicated time points after AP washout (mean ± SEM, n = 3, >10 cells/experiment). (C) Total cell fluorescence levels of GT-FM and giantin were determined using ImageJ and normalized to the level of AP treated cells before washout (mean ± SEM, n = 3, >10 cells/experiment). (D, E) Blot and quantification showing level of GT-FM in cell extracts prepared at the indicated times after AP washout with GPP130 as a loading control (mean ± SEM, n = 3).

Mentions: Because both Man1 and GPP130 are predominately in the cis-Golgi at steady state, we next tested whether a trans-Golgi protein would also respond to oligomerization by leaving the Golgi and undergoing degradation. A similar strategy was used in which the cytoplasmic, transmembrane, and stem domains of the trans-Golgi protein galactosyltransferase (GT) were fused to the tandem array of FM domains followed, in this case, by GFP. Before AP washout, the construct was strongly Golgi localized, but after washout, it selectively redistributed to peripheral punctae, and, over time, the total GT-FM levels per cell were reduced (Figure 9A). These changes were marked compared with giantin in the same cells, and both appearance in punctae (Figure 9B) and selective loss (Figure 9C) were confirmed by image analysis. As expected, degradation was apparent by immunoblot analysis (Figure 9, D and E). Cycloheximide treatment showed that all of our constructs (GPP130-FM, Man1-FM, and GT-GM) were stable in the Golgi in their nonoligomerized forms, with redistribution and degradation occurring only upon oligomerization induced by AP washout (Supplemental Figure S1).


Induced oligomerization targets Golgi proteins for degradation in lysosomes.

Tewari R, Bachert C, Linstedt AD - Mol. Biol. Cell (2015)

Induced oligomerization causes degradation of galactosyltransferase. (A) Images show thresholded GFP fluorescence of GT-FM and anti-giantin antibody staining after the indicated times of AP washout. Bar, 5 μm. (B) Quantified number of cytoplasmic punctae per cell is shown for GT-FM and the giantin control at the indicated time points after AP washout (mean ± SEM, n = 3, >10 cells/experiment). (C) Total cell fluorescence levels of GT-FM and giantin were determined using ImageJ and normalized to the level of AP treated cells before washout (mean ± SEM, n = 3, >10 cells/experiment). (D, E) Blot and quantification showing level of GT-FM in cell extracts prepared at the indicated times after AP washout with GPP130 as a loading control (mean ± SEM, n = 3).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4666137&req=5

Figure 9: Induced oligomerization causes degradation of galactosyltransferase. (A) Images show thresholded GFP fluorescence of GT-FM and anti-giantin antibody staining after the indicated times of AP washout. Bar, 5 μm. (B) Quantified number of cytoplasmic punctae per cell is shown for GT-FM and the giantin control at the indicated time points after AP washout (mean ± SEM, n = 3, >10 cells/experiment). (C) Total cell fluorescence levels of GT-FM and giantin were determined using ImageJ and normalized to the level of AP treated cells before washout (mean ± SEM, n = 3, >10 cells/experiment). (D, E) Blot and quantification showing level of GT-FM in cell extracts prepared at the indicated times after AP washout with GPP130 as a loading control (mean ± SEM, n = 3).
Mentions: Because both Man1 and GPP130 are predominately in the cis-Golgi at steady state, we next tested whether a trans-Golgi protein would also respond to oligomerization by leaving the Golgi and undergoing degradation. A similar strategy was used in which the cytoplasmic, transmembrane, and stem domains of the trans-Golgi protein galactosyltransferase (GT) were fused to the tandem array of FM domains followed, in this case, by GFP. Before AP washout, the construct was strongly Golgi localized, but after washout, it selectively redistributed to peripheral punctae, and, over time, the total GT-FM levels per cell were reduced (Figure 9A). These changes were marked compared with giantin in the same cells, and both appearance in punctae (Figure 9B) and selective loss (Figure 9C) were confirmed by image analysis. As expected, degradation was apparent by immunoblot analysis (Figure 9, D and E). Cycloheximide treatment showed that all of our constructs (GPP130-FM, Man1-FM, and GT-GM) were stable in the Golgi in their nonoligomerized forms, with redistribution and degradation occurring only upon oligomerization induced by AP washout (Supplemental Figure S1).

Bottom Line: These were targeted to the cis- and trans-Golgi, respectively, using domains from mannosidase-1 and galactosyltransferase.Significantly, upon oligomerization, each redistributed to peripheral punctae and was degraded.This occurred in the absence of detectable UPR activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213.

No MeSH data available.


Related in: MedlinePlus