1 1Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6082, USA
3 Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6082, USA
5 Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6082, USA
6 Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6082, USA
2 Integrated Imaging Center, Department of Biology, Johns Hopkins University, Baltimore, MD 21218, USA
4 Institut Curie, CNRS-UMR144, F-75248 Paris, Cedex 75005, France
* Author for correspondence (e-mail: marksm{at}mail.med.upenn.edu)
Accepted 2 March 2005
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Dynein, Dynactin, Centrosome, Retrograde transport, Endosome
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
As part of an effort to understand membrane dynamics at the TGN, we have been studying members of a group of peripheral TGN membrane proteins, referred to here as GRIP proteins, characterized by extensive predicted coiled-coil structure and a C-terminal GRIP (golgin-97, RanBP1, Imh1p, p230) domain (Barr, 1999; Kjer-Nielsen et al., 1999a
; Munro and Nichols, 1999
). Two mammalian GRIP proteins, tGolgin-1 (also known as golgin-245 and trans-Golgi p230; gene name, golga4) (Cowan et al., 2002
; Erlich et al., 1996
; Fritzler et al., 1995
) and golgin-97 (gene name, golga1) (Griffith et al., 1997
), were identified as autoantigens in patients with Sjögren's syndrome. Two others, GCC88 and GCC185 (Luke et al., 2003
), were identified by sequence homology and appear to be functionally unrelated (Derby et al., 2004
). The GRIP domains of tGolgin-1 and golgin-97 mediate localization to the cytoplasmic face of the TGN via interaction with the small GTPase Arl1 (Lu and Hong, 2003
; Panic et al., 2003a
; Panic et al., 2003b
; Setty et al., 2003
; Wu et al., 2004
). Although modest production of these isolated GRIP domains results in the displacement of endogenous golgin-97 and tGolgin-1 from the TGN (Kjer-Nielsen et al., 1999a
; Kjer-Nielsen et al., 1999b
), probably by competition for limiting Arl1-GTP, overproduction of these GRIP domains further results in extensive morphological disruption of the TGN concomitant with an inhibition of retrograde transport of TGN-resident proteins from endosomes (Yoshino et al., 2003
). This result suggested that tGolgin-1, golgin-97 and/or other Arl1 effectors function in regulating vesicular transport from endosomes to the TGN, a notion supported by genetic analyses of the single GRIP protein Imh1p in yeast (Li and Warner, 1996
; Siniossoglou et al., 2000
; Tsukada et al., 1999
) and recently demonstrated for mammalian golgin-97 (Lu et al., 2004
). To test whether tGolgin-1 shares a similar function, we assessed the consequences of reduced tGolgin-1 production in HeLa cells. Our results are consistent with a role for tGolgin-1 in regulating retrograde transport of Shiga toxin to the Golgi. More surprisingly, however, our results demonstrate a critical requirement for tGolgin-1 production in the minus-end-directed transport of Golgi elements along microtubules. Based on our data and the reported Golgi fragmentation observed upon disruption of other proteins required for retrograde transport (Lu et al., 2004
; Seaman, 2004
), we propose that recruitment of microtubule motors to the Golgi requires tGolgin-1-dependent retrograde transport from endosomes.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The following antibodies against the indicated proteins were obtained from the sources listed: sheep anti-TGN46, Serotec, Oxford, UK; rabbit anti-golgin-97 and monoclonal antibody (mAb) anti-golgin-97, E. K. L. Chan (Scripps Research Institute, La Jolla, CA) (Griffith et al., 1997); mAb anti-giantin, H.-P. Hauri (University of Basel, Switzerland) (Linstedt and Hauri, 1993
); rabbit anti-ß-1,4-galactosyl transferase (GalT), E. Berger (University of Zurich, Switzerland) (Berger and Hesford, 1985
); mAb anti-Lamp1 (H4A3), Developmental Studies Hybridoma Bank, Iowa City, IA; anti-CD63 (mAb against CD63), Beckman Coulter, Fullerton, CA; anti-tubulin (mAb YL1/2), Accurate Chemical, Westbury, NY; anti-transferrin receptor (mAb B3/25), Roche Molecular Biochemicals, Indianapolis, IN. mAbs against human tGolgin-1 (p230), EEA1, GM130, p115 and GS28 were from Becton Dickinson/Pharmingen (San Diego, CA). mAbs against acetylated tubulin,
-tubulin and
-tubulin were from Sigma (St Louis, MO). Secondary antibodies against mouse, rabbit, sheep or rat IgG and conjugated to rhodamine red X (RRX), fluorescein isothiocyanate (FITC) or aminomethylcoumarin acetate (AMCA) were from Jackson ImmunoResearch (West Grove, PA).
|
|
Cell culture and transfection
HeLa cells were maintained in Dulbecco's modified Eagle's medium with 10% foetal bovine serum (FBS). For siRNA transduction, 0.75x105 to 3x105 HeLa cells per well were plated in 12-well plates in fresh medium without antibiotics. Cells were transduced on day 2 with 1.68 µg siRNA using Oligofectamine (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. In most experiments, the transfection was repeated 24 hours later, cells were replated onto coverslips the next day, then fixed 1 day later and analysed. Co-transfection of plasmid DNA (0.5-2 µg per well) and siRNA (0.84 µg per well) was done using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions in a single transfection. Cells were replated on coverslips the next day and fixed 2 days after transfection. Transfection of plasmids was done in six-well dishes using calcium-phosphate precipitation (Marks et al., 1996) with 7.0-7.5 µg total DNA or in 12-well dishes using Lipofectamine 2000 (Santini et al., 1998
) with 2 µg total DNA, and cells were analysed 2 days after transfection. HeLa cells stably producing GRASP65-EGFP (HeLa-GRASP65-EGFP) were prepared by transfection using calcium-phosphate precipitation and selection in 0.8 mg ml1 geneticin (G418). G418-resistant cells were segregated and screened for GFP production by flow cytometry using a FACStar Plus cell sorter (BD Biosciences, San Jose, CA), cloned by limiting dilution and screened for GRASP65-EGFP Golgi localization by fluorescence microscopy.
Nocodazole treatment
To depolymerize microtubules, HeLa cells were incubated on ice for 15 minutes and then treated with 5 µg ml1 nocodazole (Sigma) in DMEM containing 25 mM HEPES, pH 7.4, 10% FBS at 37°C for 2 hours, followed by fixation. Staining with anti--tubulin antibodies showed only a diffuse cytoplasmic stain by immunofluorescence microscopy (IFM).
Brefeldin-A washout at 16°C
HeLa cells were incubated with 1 µg ml1 brefeldin A in DMEM containing 10% FBS for 1 hour, washed twice with PBS, incubated with DMEM containing 10% FBS, 25 mM HEPES, pH 7.4, at 16°C for 3 hours and then incubated at 37°C as indicated. At the end of each time period, cells were fixed and analysed by fluorescence microscopy.
Internalization and post-endocytic transport of Shiga-toxin B fragment
HeLa cells were incubated on ice for 45 minutes with Alexa-488-conjugated Shiga-toxin B fragment (STxB, 1:100 dilution of concentrated stock). Cells were washed with PBS, chased at 37°C for 60 minutes and then fixed and analysed by fluorescence microscopy. Some cells were pretreated on ice for 15 minutes and 37°C for 2 hours with nocodazole (5 µg ml1) before STxB incorporation in the presence of nocodazole (1 µg ml1). Cells transduced with siRNA were used 3 days after the first transfection.
Immunofluorescence microscopy
HeLa cells were fixed with either 2% formaldehyde for 25 minutes at room temperature essentially as described (Marks et al., 1995) or with methanol at 20°C for 5 minutes followed by immunostaining. Cells were analysed on a Leica Microsystems (Bannockburn, IL) DM IRBE microscope using a Hamamatsu (Hamamatsu, Japan) Orca digital camera and Improvision OpenLab software (Lexington, MA). Most images shown are compressed from multiple deconvolved layers from a z-axis series of images taken at 0.2 µm increments. Quantitation of overlap for STxB and Golgi markers was done on non-deconvolved images using the Density Slicing and Quantitation modules of the OpenLab software.
Video confocal microscopy
HeLa-GRASP65-EGFP were grown on glass-bottomed microwell dishes (MatTek, Ashland, MA). For controls, GRASP65-EGFP was accumulated at ER exit sites by treating cells as described above for brefeldin-A washout at 16°C. For nocodazole treatment, HeLa-GRASP65-EGFP were incubated on ice for 15 minutes followed by incubation with 5 µg ml1 nocodazole at 37°C for 2 hours and during the analysis. For dynamitin overproduction and tGolgin-1 depletion, cells were transduced with plasmid or siRNA as described above and observed 2 days after transfection. Time-lapse z-axis series images (0.5 µm steps) were analysed at intervals of 20 seconds (control) or 60 seconds (all others) in a 37°C heating chamber on a Ultraview LCI Nipkow disc confocal microscope (Perkin-Elmer, Norwalk, CT) attached to a Nikon TE300 inverted microscope (Nikon, Melville, NY) fitted with a 60x oil-immersion objective, as described (Whiteman et al., 2003) or on a Leica DM IRBE microscope using a Hamamatsu Orca digital camera. Captured images were analysed using Improvision OpenLab software (Lexington, MA). The distance that each mobile Golgi element moved per minute in each condition was assessed by comparing sequential images using OpenLab software. Statistical analyses used Stat2000.xls (S. Yamazaki) and Microsoft Excel software.
Electron microscopy
HeLa cells transduced twice with siRNA-1 or control siRNA were split into 10 cm dishes and fixed 2 days after the second transfection using 1.5% glutaraldehyde, 3% formaldehyde as described (McCaffery and Farquhar, 1995; Yoshino et al., 2003
). Parallel analyses by IFM indicated that 85% of the cells transduced with siRNA-1 had undetectable levels of tGolgin-1 production. Cells were osmicated, embedded in epon, sectioned and analysed on a Philips 420 transmission electron microscope as described (McCaffery and Farquhar, 1995
; Yoshino et al., 2003
). Morphometry was performed on random images of phenotypic cells from the siRNA-1 sample and of random cells in the control sample using analySIS v3.2 software (Soft Imaging System, Lakewood, CO).
Immunoblotting
Immunoblotting of Triton-X-100 cell lysates fractionated by SDS-PAGE on 7.5% acrylamide gels was performed essentially as described (Berson et al., 2003) using mouse anti-p230 antibodies, enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Piscataway, NJ) and analysis on a Molecular Dynamics Storm 860 PhosphorImager (Amersham Pharmacia Biotech). As loading controls, blots were probed with anti-tubulin antibodies.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
TGN46 dispersal has also been observed in cells overproducing dominant-negative GRIP domains from tGolgin-1 and golgin-97 (Yoshino et al., 2003). However, the phenotype of tGolgin-1-depleted cells differed in several respects. First, whereas both tGolgin-1 and golgin-97 were displaced to the cytosol in GRIP-domain-overproducing cells, golgin-97 in tGolgin-1-depleted cells was localized to the peripheral TGN46-containing membrane elements (Fig. 3C,D). Second, unlike in cells overproducing GRIP domains, markers of the Golgi stack (e.g. giantin, GM130 and GS28; and also, not shown, GalT, p115 and GRASP65) were also redistributed to peripheral puncta, as observed by IFM (Fig. 3E-K). Puncta labelled for Golgi markers partially overlapped those labelled by TGN46 in a polarized manner in deconvolved images of z-axis sections (Fig. 3I-K), suggesting that Golgi cis
trans polarity was maintained. Electron-microscopic analysis of these cells indicated that the normally centralized Golgi complex found in untransduced cells or cells transduced with a control siRNA (Fig. 3L) was replaced by multiple `mini stacks' (Fig. 3M-Q; see also Fig. 8) that were present predominantly, but not exclusively, at the cell periphery. These mini stacks were morphologically similar to intact Golgi stacks in control cells but were more numerous and each one comprised a smaller total area, as determined by morphometry [0.20±0.10 µm2 per stack (n=31) vs 0.61±0.38 µm2 per stack (n=15) for cells transduced with siRNA-1 vs control siRNA, respectively]. In contrast to Golgi and TGN residents, the distribution of resident proteins of other compartments, including ER, early endosomes, recycling endosomes, late endosomes and lysosomes, was not affected by tGolgin-1 depletion (see Fig. S1 in supplementary material; however, see Fig. 8 regarding the morphology of late endosomes and lysosomes), and the uptake and intracellular accumulation of transferrin was not perceptibly altered (data not shown). These data indicate that the Golgi/TGN was specifically decentralized in cells depleted of tGolgin-1.
|
|
First, parallel staining for Golgi and/or TGN markers and microtubules showed that, as in dynamitin-overproducing cells, microtubules were intact and the Golgi-like elements in tGolgin-1-depleted cells appeared to be aligned along them (Fig. 4A-F). Second, using -tubulin as a marker for the MTOC, we asked whether Golgi elements remained clustered near the MTOC as they do in untreated cells (Fig. 4G-I). Although the Golgi was largely dispersed to peripheral stacks in cells treated with nocodazole, large clusters of Golgi remnants harbouring GalT-EGFP were colocalized with
-tubulin (Fig. 4J-L), suggesting that a significant population of Golgi fragments remained associated with nocodazole-insensitive `stubs' of microtubules at the MTOC. By contrast, in cells that either overproduced dynamitin-p50 (Fig. 4M-O) or were depleted of tGolgin-1 by siRNA-1 (Fig. 4P-R), no such remnants accumulated near the MTOC. This difference between nocodazole-treated and either dynamitin-overproducing or tGolgin-1-depleted cells was particularly evident using giantin as a marker (compare Fig. 3J with Fig. 4S-X). The data support the notion that nocodazole treatment and dynamitin overproduction distinguish two phases in Golgi accumulation at the MTOC, a transport phase (dependent on dynein-dynactin) and a subsequent tethering phase. Importantly, tGolgin-1 depletion mimics dynamitin overproduction, suggesting that tGolgin-1 production might be required for the transport phase of Golgi elements toward the minus end of microtubules.
To determine the proximity of Golgi-like elements to ER exit sites in tGolgin-1-depleted cells, the redistributed Golgi and TGN markers were compared with the distribution of Sec13-EGFP (an EGFP-conjugated subunit of the COPII coat complex that marks these sites) (Hammond and Glick, 2000). In cells co-transfected with low levels of Sec13-EGFP and siRNA-1 (Fig. 5A-C) or treated with nocodazole (Hammond and Glick, 2000
) (Fig. 5D-F), TGN46- and giantin-labelled vesicles cluster around elements labelled by Sec13-EGFP, indicating that Golgi/TGN elements indeed accumulate near ER exit sites in these cells.
|
Finally, we analysed the effects of tGolgin-1 depletion on Golgi element motility using video microscopy. An EGFP-tagged form of the Golgi matrix protein GRASP65 (Lane et al., 2002) was stably produced in HeLa cells and individual clones were chosen that had predominant Golgi localization by fluorescence microscopy. The cells were then analysed by video microscopy after treatment with nocodazole or after transduction with siRNA-1 or dynamitin expression vector, and summary images of representative videos were prepared in which EGFP signal in the initial frame is coloured white and subsequent frames are coloured red (Fig. 5M-O). The results demonstrate that GRASP65-containing elements in all three sets of cells were relatively immobile. As a control, we performed similar analyses of cells in the process of recovering at 16°C from brefeldin-A treatment. Although some of the larger elements in these cells were also relatively immobile during the course of the experiment, the majority of them and most of the smaller elements were highly motile (Fig. 5P). Quantitation of the movement of random individual elements from all experimental samples (Table 2, Fig. 5Q) confirms that both the proportion of motile elements and the median rate of movement were similar in siRNA-1-treated and dynamitin-overproducing cells, much lower than in control cells and slightly higher than in nocodazole-treated cells. Taken together, these data show that the Golgi elements formed in the presence of siRNA-1 are relatively immobile and similar to those that form in the absence of dynein-dynactin motor function. The results are consistent with a requirement for tGolgin-1 in minus-end-directed motility of Golgi elements along microtubules rather than in the retention or tethering of motile Golgi elements at the MTOC.
|
|
|
HeLa cells were incubated with Alexa-488-conjugated STxB at 0°C for 45 minutes, washed and then warmed to 37°C for 60 minutes to permit internalization of the bound STxB. In control cells, a large proportion of the STxB at this time point colocalized with GM130 at the Golgi (Fig. 7A-C). By contrast, a much smaller proportion of STxB colocalized with GM130 in dispersed Golgi structures in cells depleted of tGolgin-1 (Fig. 7D-F; notice that at no time was significant colocalization of STxB with TGN46 observed in data not shown). Quantitation of the proportion of the total GM130-containing cell area that also contained STxB showed at least a halving of the ability of STxB to reach the Golgi in tGolgin-1-depleted cells (Table 3); this is probably an underestimate of the defect based on the method of quantitation used. The defect in transport was not an indirect effect of Golgi dispersal, because STxB localized efficiently to GM130-containing puncta in control cells treated with nocodazole (Fig. 7G-I, Table 3). The decrease in STxB transport to Golgi elements was compensated by an increased colocalization with early endosomal markers transferrin receptor and EEA1 (see Fig. S3 in supplementary material) and, importantly, a dramatic increase in colocalization with perinuclear late endosomes marked by Lamp1 (Fig. 7J-O). These data indicate that, in the absence of tGolgin-1, STxB is inefficiently distributed to the Golgi from early endosomes and is significantly mis-sorted to late endosomes.
|
STxB trafficking probably reflects the intracellular itinerary of its glycosphingolipid ligand (Sandvig and van Deurs, 2002), and indeed we observed similar lysosomal mis-sorting of an internalized synthetic glycosphingolipid analogue, N-[5-(5,7-dimethyl BODIPY)-1pentanoyl]-lactosylceramide (data not shown), although the effect was difficult to quantify. If tGolgin-1 was required for post-endocytic sorting of glycolipids then cells lacking tGolgin-1 would be predicted to accumulate unusual late endosomal membrane structures owing to the increased content of glycolipids and their associated cholesterol, as is observed in cells from patients with lysosomal lipid-storage diseases (Marks and Pagano, 2002
). Consistent with this prediction, ultrastructural analysis of tGolgin-1-depleted cells revealed an abundance of unusual large vacuolar structures with clumps of internal membranes in `whorls' (Fig. 8A,B) or vesicle-like profiles (Fig. 8C), much like those found in lipid-storage-disease patients (Marks and Pagano, 2002
). Quantification showed that whorls were found in seven out of 123 random images from tGolgin-1-depleted cells, but none out of 51 random images of cells treated with a control siRNA. Although the morphology of these compartments was altered, the distribution of most lysosomal proteins to them was not adversely affected (see Fig. S1 in supplementary material). These data support the interpretation that internalized glycosphingolipids are significantly mis-sorted to late endosomes/lysosomes in cells depleted of tGolgin-1, and implicate tGolgin-1 in regulating the retrograde transport of glycolipids.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
`Dispersal' of Golgi-resident proteins, as observed by fluorescence microscopy, can result from any number of insults to the Golgi complex, including disruption of vesicular traffic, cisternal stacking, organelle motility or tethering to the MTOC (Warren and Shorter, 2002). Our data indicate that tGolgin-1 production is required specifically for the movement of Golgi or pre-Golgi elements toward the MTOC. First, the microtubule network in cells lacking tGolgin-1 was intact and Golgi elements appeared to be associated with them, ruling out any gross effects on microtubule organization or Golgi-associated microtubule-binding proteins, as might be predicted from the observed in vitro interactions of tGolgin-1 with the actin/microtubule-binding protein ACF7/MACF1 (Kakinuma et al., 2004
). Second, unlike depletion of the Golgi-vesicle tethering factor golgin-84 (Diao et al., 2003
; Malsam et al., 2005
), depletion of tGolgin-1 did not affect anterograde transport of VSV-G/EGFP. Third, the Golgi dispersal observed upon tGolgin-1 depletion mimics that observed upon interfering with the dynein-dynactin complex or microtubules (Burkhardt et al., 1997
; Cole et al., 1996a
; Hammond and Glick, 2000
) in that polarized Golgi mini stacks accumulate at ER exit sites, the Golgi stacks remain functional for anterograde transport and motility is inhibited. Moreover, unlike in cells treated acutely with nocodazole in which dynein-dynactin function is intact and microtubules only transiently disrupted, tGolgin-1-depleted cells showed no Golgi fragments tethered at microtubule `stubs' at the MTOC. Fourth, rapid bidirectional movement of Golgi elements along microtubules, as observed for untethered melanosomes in cells lacking Rab27a (Wu et al., 2001
), was not observed in tGolgin-1-depleted cells, indicating a defect in motility that precedes tethering. Although we did not detect a quantitative decrease in dynein or dynactin subunits in pelletable fractions of HeLa cell homogenates upon tGolgin-1 depletion, as might be predicted if dynein-dynactin recruitment was inhibited, the minimal steady-state accumulation of these subunits at the Golgi in untreated cells (data not shown) renders this result uninterpretable and indicates that dynein-dynactin complexes associate dynamically with MTOC-bound pre-Golgi membranes in a manner that is not reflected by steady-state accumulation. We infer from the distinct phenotypes of chronic dynein-dynactin disruption (by dynamitin-p50 overproduction or tGolgin-1 depletion) and acute microtubule disruption (by nocodazole treatment) that tGolgin-1 function is required for dynamic association of Golgi membranes with an active motor complex.
How is tGolgin-1 function in Golgi migration exerted? The absence of tGolgin-1 from actively migrating Golgi/pre-Golgi elements bound for the MTOC in control cells indicates that tGolgin-1 is not a component of a receptor or activator for dynein-dynactin complexes on the cytoplasmic face of these elements. The unlikely possibility that a small, undetectable fraction of tGolgin-1 on these elements directly binds to dynein or dynactin is difficult to reconcile with the TGN localization of tGolgin-1 at steady state (Gleeson et al., 1996) and with our inability to detect interactions between tGolgin-1 and dynein or dynactin subunits using a range of biochemical approaches (data not shown). An indirect requirement for tGolgin-1 in Golgi motility is further supported by the failure to observe Golgi dispersal upon microinjection of antibodies against tGolgin-1 into HeLa cells, which resulted in a transient loss of detectable production of the endogenous protein (data not shown). Moreover, the heterogeneity of the response (50-65% of tGolgin-1-depleted cells showed Golgi dispersal) is inconsistent with a direct role. The proportion of tGolgin-1-depleted cells with Golgi dispersal was not dependent on time after transfection and was unchanged by inhibition of cell-cycle progression or by recovery after brefeldin-A treatment (data not shown), ruling out a requirement for mitotic or drug-induced Golgi dispersal before the tGolgin-1 requirement. We therefore favour a model in which tGolgin-1 acts indirectly by regulating the transient localization or activation of a crucial dynein-dynactin recruitment or activation factor.
How might tGolgin-1 regulate such a factor? We have shown here that cells depleted of tGolgin-1 mis-sort the internalized glycolipid ligand STxB to late endosomes/lysosomes and accumulate aberrant endosomal structures. These results imply that Golgi redistribution in tGolgin-1-depleted cells might be a secondary consequence of disrupting membrane recycling. This is consistent with the function of another GRIP-domain protein golgin-97 (Lu et al., 2004) and with results of dominant-negative GRIP-domain overproduction, in which endosome-to-TGN recycling was inhibited (Yoshino et al., 2003
). Indeed, although depletion of some retrograde transport factors has no effect on Golgi morphology (Saint-Pol et al., 2004
), several groups have recently reported Golgi dispersal of an undefined nature after siRNA-mediated depletion of other factors regulating endosome-to-TGN transport, including retromer components (Seaman, 2004
) and golgin-97 (Lu et al., 2004
). We suggest that retrograde transport is required to localize membrane-associated factors that function directly in dynein-dynactin recruitment to pre-Golgi elements. Such factors might be either integral membrane proteins or lipids, both of which have been shown to cycle from endosomes to the Golgi (Ghosh et al., 1998
; Johannes and Goud, 1998
; Mallet and Maxfield, 1999
), including to the cis Golgi (Natarajan and Linstedt, 2004
), and might tether known dynein-dynactin recruitment proteins such as Bicaudal-D (Matanis et al., 2002
; Short et al., 2002
), ßIII spectrin (Holleran et al., 2001
) and CLIPR-59 (Perez et al., 2002
). An intriguing possibility is that the relevant targets of retrograde transport are glycolipids and/or cholesterol. A consequence of chronic mis-sorting to late endosomes would be eventual depletion from the Golgi of recycling glycosphingolipids and probably concomitant depletion of associated cholesterol (Brown, 1998
; Marks and Pagano, 2002
; van Meer, 2002
). Although glycosphingolipid deficiency per se does not result in Golgi dispersal (Sprong et al., 2001
), depletion or enhancement of Golgi cholesterol is known to affect Golgi distribution and intra-Golgi transport (Stüven et al., 2003
), as well as the requirement for the kinesin family member KIFC3 in Golgi motility (Xu et al., 2002
). Cell-to-cell variation in cholesterol or glycosphingolipid content might explain why only 50-65% of tGolgin-1-depleted cells show Golgi dispersal, and cholesterol depletion might explain the reduced steady-state localization of a glycosylphosphatidylinositol-linked protein to the plasma membrane in cells overproducing a tGolgin-1-derived peptide (Kakinuma et al., 2004
).
The localization of internalized STxB to the Golgi was impaired but not ablated in tGolgin-1-depleted cells, indicating that tGolgin-1 production facilitates but is not required for retrograde transport. Indeed, tGolgin-1 might influence retrograde transport indirectly. The mis-sorting of STxB to late endosomes is distinct from the early-endosome accumulation observed upon direct inhibition of the endosome-to-TGN pathway (Mallard et al., 1998; Mallard et al., 2002
; Saint-Pol et al., 2004
; Tai et al., 2004
) but similar to that observed for STxB from detergent-soluble lipid microdomains of macrophages (Falguières et al., 2001
) and for a subset of glycosphingolipids in fibroblasts from patients with sphingolipid-storage diseases (Marks and Pagano, 2002
). One possibility, consistent with budding of tGolgin-1-enriched vesicles from Golgi membranes in vitro (Brown et al., 2001
; Gleeson et al., 1996
) and the apparent effect of a tGolgin-1 peptide on plasma-membrane accumulation of a glycosylphosphatidylinositol-linked marker (Kakinuma et al., 2004
), is that tGolgin-1 participates in anterograde transport of a subset of cargo that might impact the overall cellular distribution of glycosphingolipids and/or cholesterol, as occurs in glycolipid-storage diseases (Marks and Pagano, 2002
). Such a role for tGolgin-1 in anterograde transport might balance a retrograde pathway regulated by golgin-97 (Lu et al., 2004
); interference with both pathways might restore the balance of lipids at the Golgi, potentially explaining why Golgi dispersal is not observed in cells depleted of Arl1 (Lu and Hong, 2003
) or overproducing GRIP domains (Yoshino et al., 2003
). Our studies thus provide a springboard for future investigations into the direct function of tGolgin-1 and the relationship between retrograde transport, lipid distribution and Golgi motility.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Barr, F. A. (1999). A novel Rab6-interacting domain defines a family of Golgi-targeted coiled-coil proteins. Curr. Biol. 9, 381-384.[CrossRef][Medline]
Berger, E. G. and Hesford, F. J. (1985). Localization of galactosyl- and sialyltransferase by immunofluorescence: evidence for different sites. Proc. Natl. Acad. Sci. USA 82, 4736-4739.
Bergmann, J. E., Tokuyasu, K. T. and Singer, S. J. (1981). Passage of an integral membrane protein, the vesicular stomatitis virus glycoprotein, through the Golgi apparatus en route to the plasma membrane. Proc. Natl. Acad. Sci. USA 78, 1746-1750.
Berson, J. F., Theos, A. C., Harper, D. C., Tenza, D., Raposo, G. and Marks, M. S. (2003). Proprotein convertase cleavage liberates a fibrillogenic fragment of a resident glycoprotein to initiate melanosome biogenesis. J. Cell Biol. 161, 521-533.
Bonifacino, J. S., Cosson, P. and Klausner, R. D. (1990). Colocalized transmembrane determinants for ER degradation and subunit assembly explain the intracellular fate of TCR chains. Cell 63, 503-513.[CrossRef][Medline]
Brown, D. L., Heimann, K., Lock, J., Kjer-Nielsen, L., van Vliet, C., Stow, J. L. and Gleeson, P. A. (2001). The GRIP domain is a specific targeting sequence for a population of trans-Golgi network derived tubulo-vesicular carriers. Traffic 2, 336-344.[CrossRef][Medline]
Brown, R. E. (1998). Sphingolipid organization in biomembranes: what physical studies of model membranes reveal. J. Cell Sci. 111, 1-9.
Burkhardt, J. K., Echeverri, C. J., Nilsson, T. and Vallee, R. B. (1997). Overexpression of the dynamitin (p50) subunit of the dynactin complex disrupts dynein-dependent maintenance of membrane organelle distribution. J. Cell Biol. 139, 469-484.
Cole, N. B., Sciaky, N., Marotta, A., Song, J. and Lippincott-Schwartz, J. (1996a). Golgi dispersal during microtubule disruption: regeneration of Golgi stacks at peripheral endoplasmic reticulum exit sites. Mol. Biol. Cell 7, 631-650.[Abstract]
Cole, N. B., Smith, C. L., Sciaky, N., Terasaki, M., Edidin, M. and Lippincott-Schwartz, J. (1996b). Diffusional mobility of Golgi proteins in membranes of living cells. Science 273, 797-801.[Abstract]
Corthesy-Theulaz, I., Pauloin, A. and Pfeffer, S. R. (1992). Cytoplasmic dynein participates in the centrosomal localization of the Golgi complex. J. Cell Biol. 118, 1333-1345.[Abstract]
Cowan, D. A., Gay, D., Bieler, B. M., Zhao, H., Yoshino, A., Davis, J. G., Tomayko, M. M., Murali, R., Greene, M. I. and Marks, M. S. (2002). Characterization of mouse tGolgin-1 (golgin-245/trans Golgi p230/256kD golgin) and its upregulation during oligodendrocyte development. DNA Cell Biol. 21, 505-517.[CrossRef][Medline]
Derby, M. C., van Vliet, C., Brown, D., Luke, M. R., Lu, L., Hong, W., Stow, J. L. and Gleeson, P. A. (2004). Mammalian GRIP domain proteins differ in their membrane binding properties and are recruited to distinct domains of the TGN. J. Cell Sci. 117, 5865-5874.
Diao, A., Rahman, D., Pappin, D. J. C., Lucocq, J. and Lowe, M. (2003). The coiled-coil membrane protein golgin-84 is a novel Rab effector required for Golgi ribbon formation. J. Cell Biol. 160, 201-212.
Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K. and Tuschl, T. (2001). Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494-498.[CrossRef][Medline]
Erlich, R., Gleeson, P. A., Campbell, P., Dietzsch, E. and Toh, B.-H. (1996). Molecular characterization of trans-Golgi p230. A human peripheral membrane protein encoded by a gene on chromosome 6p12-22 contains extensive coiled-coil -helical domains and a granin motif. J. Biol. Chem. 271, 8328-8337.
Falguières, T., Mallard, F., Baron, C., Hanau, D., Lingwood, C., Goud, B., Salamero, J. and Johannes, L. (2001). Targeting of Shiga toxin B-subunit to retrograde transport route in association with detergent-resistant membranes. Mol. Biol. Cell 12, 2453-2468.
Fritzler, M. J., Lung, C.-C., Hamel, J. C., Griffith, K. J. and Chan, E. K. L. (1995). Molecular characterization of golgin-245, a novel Golgi complex protein containing a granin signature. J. Biol. Chem. 270, 31262-31268.
Ghosh, R. N., Mallet, W. G., Soe, T. T., McGraw, T. E. and Maxfield, F. R. (1998). An endocytosed TGN38 chimeric protein is delivered to the TGN after trafficking through the endocytic recycling compartment in CHO cells. J. Cell Biol. 142, 923-936.
Gillingham, A. K., Tong, A. H. Y., Boone, C. and Munro, S. (2004). The GTPase Arf1p and the ER to Golgi cargo receptor Erv14p cooperate to recruit the golgin Rud3p to the cis-Golgi. J. Cell Biol. 167, 281-292.
Gleeson, P. A., Anderson, T. J., Stow, J. L., Griffiths, G., Toh, B. H. and Matheson, F. (1996). p230 is associated with vesicles budding from the trans-Golgi network. J. Cell Sci. 109, 2811-2821.
Griffith, K. J., Chan, E. K., Lung, C. C., Hamel, J. C., Guo, X., Miyachi, K. and Fritzler, M. J. (1997). Molecular cloning of a novel 97-kD Golgi complex autoantigen associated with Sjogren's syndrome. Arthritis Rheum. 40, 1693-1702.[Medline]
Hammond, A. T. and Glick, B. S. (2000). Dynamics of transitional endoplasmic reticulum sites in vertebrate cells. Mol. Biol. Cell 11, 3013-3030.
Harada, A., Takei, Y., Kanai, Y., Tanaka, Y., Nonaka, S. and Hirokawa, N. (1998). Golgi vesiculation and lysosome dispersion in cells lacking cytoplasmic dynein. J. Cell Biol. 141, 51-59.
Ho, W. C., Allan, V. J., van Meer, G., Berger, E. G. and Kreis, T. E. (1989). Reclustering of scattered Golgi elements occurs along microtubules. Eur. J. Cell Biol. 48, 250-263.[Medline]
Holleran, E. A., Ligon, L. A., Tokito, M., Stankewich, M. C., Morrow, J. S. and Holzbaur, E. L. F. (2001). ßIII spectrin binds to the Arp1 subunit of dynactin. J. Biol. Chem. 276, 36598-36605.
Infante, C., Ramos-Morales, F., Fedriani, C., Bornens, M. and Rios, R. M. (1999). GMAP-210, a cis-Golgi network-associated protein, is a minus end microtubule-binding protein. J. Cell Biol. 145, 83-98.
Johannes, L. and Goud, B. (1998). Surfing on a retrograde wave: how does Shiga toxin reach the endoplasmic reticulum? Trends Cell Biol. 8, 158-162.[CrossRef][Medline]
Kakinuma, T., Ichikawa, H., Tsukada, Y., Nakamura, T. and Toh, B. H. (2004). Interaction between p230 and MACF1 is associated with transport of a glycosyl phosphatidyl inositol-anchored protein from the Golgi to the cell periphery. Exp. Cell Res. 298, 388-398.[CrossRef][Medline]
Kjer-Nielsen, L., Teasdale, R. D., van Vliet, C. and Gleeson, P. A. (1999a). A novel Golgi-localisation domain shared by a class of coiled-coil peripheral membrane proteins. Curr. Biol. 9, 385-388.[CrossRef][Medline]
Kjer-Nielsen, L., van Vliet, C., Erlich, R., Toh, B.-H. and Gleeson, P. A. (1999b). The Golgi targeting sequence of the peripheral membrane protein p230. J. Cell Sci. 112, 1645-1654.
Ladinsky, M. S., Mastronarde, D. N., McIntosh, J. R., Howell, K. E. and Staehelin, L. A. (1999). Golgi structure in three dimensions: functional insights from the Normal Rat Kidney cell. J. Cell Biol. 144, 1135-1149.
Lane, J. D., Lucocq, J., Pryde, J., Barr, F. A., Woodman, P. G., Allan, V. J. and Lowe, M. (2002). Caspase-mediated cleavage of the stacking protein GRASP65 is required for Golgi fragmentation during apoptosis. J. Cell Biol. 156, 495-509.
Li, B. and Warner, J. R. (1996). Mutation of the Rab6 homologue of Saccharomyces cerevisiae, YPT6, inhibits both early Golgi function and ribosome biosynthesis. J. Biol. Chem. 271, 16813-16819.
Linstedt, A. D. and Hauri, H. P. (1993). Giantin, a novel conserved Golgi membrane protein containing a cytoplasmic domain of at least 350 kDa. Mol. Biol. Cell 4, 679-693.[Abstract]
Lu, L. and Hong, W. (2003). Interaction of Arl1-GTP with GRIP domains recruits autoantigens Golgin-97 and golgin-245/p230 onto the Golgi. Mol. Biol. Cell 14, 3767-3781.
Lu, L., Tai, G. and Hong, W. (2004). Autoantigen golgin-97, an effector of Arl1 GTPase, participates in traffic from the endosome to the TGN. Mol. Biol. Cell 15, 4426-4443.
Luke, M. R., Kjer-Nielsen, L., Brown, D. L., Stow, J. L. and Gleeson, P. A. (2003). GRIP domain-mediated targeting of two new coiled coil proteins, GCC88 and GCC185, to subcompartments of the trans-Golgi network. J. Biol. Chem. 278, 4216-4226.
Mallard, F., Antony, C., Tenza, D., Salamero, J., Goud, B. and Johannes, L. (1998). Direct pathway from early/recycling endosomes to the Golgi apparatus revealed through the study of Shiga toxin B-fragment transport. J. Cell Biol. 143, 973-990.
Mallard, F., Tang, B. L., Galli, T., Tenza, D., Saint-Pol, A., Yue, X., Antony, C., Hong, W., Goud, B. and Johannes, L. (2002). Early/recycling endosomes-to-TGN transport involves two SNARE complexes and a Rab6 isoform. J. Cell Biol. 156, 653-664.
Mallet, W. G. and Maxfield, F. R. (1999). Chimeric forms of furin and TGN38 are transported with the plasma membrane in the trans-Golgi network via distinct endosomal pathways. J. Cell Biol. 146, 345-359.
Malsam, J., Satoh, A., Pelletier, L. and Warren, G. (2005). Golgin tethers define subpopulations of COPI vesicles. Science 307, 1095-1098.
Marks, D. L. and Pagano, R. E. (2002). Endocytosis and sorting of glycosphingolipids in sphingolipid storage disease. Trends Cell Biol. 12, 605-613.[CrossRef][Medline]
Marks, M. S., Roche, P. A., van Donselaar, E., Woodruff, L., Peters, P. J. and Bonifacino, J. S. (1995). A lysosomal targeting signal in the cytoplasmic tail of the ß chain directs HLA-DM to the MHC class II compartments. J. Cell Biol. 131, 351-369.[Abstract]
Marks, M. S., Woodruff, L., Ohno, H. and Bonifacino, J. S. (1996). Protein targeting by tyrosine- and di-leucine-based signals: evidence for distinct saturable components. J. Cell Biol. 135, 341-354.[Abstract]
Marsh, B. J., Mastronarde, D. N., Buttle, K. F., Howell, K. E. and McIntosh, R. J. (2001). Organellar relationships in the Golgi region of the pancreatic beta cell line, HIT-T15, visualized by high resolution electron tomography. Proc. Natl. Acad. Sci. USA 98, 2399-2406.
Matanis, T., Akhmanova, A., Wulf, P., del Nery, E., Weide, T., Stepanova, T., Galjart, N., Grosveld, F., Goud, B., de Zeeuw, C. I. et al. (2002). Bicaudal-D regulates COPI-independent Golgi-ER transport by recruiting the dynein-dynactin motor complex. Nat. Cell Biol. 4, 986-992.[CrossRef][Medline]
McCaffery, J. M. and Farquhar, M. G. (1995). Localization of GTPases (GTP-binding proteins) by indirect immunofluorescence and immunoelectron microscopy. Methods Enzymol. 257, 259-279.[Medline]
Munro, S. and Nichols, B. J. (1999). The GRIP domain a novel Golgi-targeting domain found in several coiled-coil proteins. Curr. Biol. 9, 377-380.[CrossRef][Medline]
Muresan, V., Stankewich, M. C., Steffen, W., Morrow, J. S., Holzbaur, E. L. and Schnapp, B. J. (2001). Dynactin-dependent, dynein-driven vesicle transport in the absence of membrane proteins: a role for spectrin and acidic phospholipids. Mol. Cell 7, 173-183.[CrossRef][Medline]
Natarajan, R. and Linstedt, A. D. (2004). A cycling cis Golgi protein mediates endosome-to-Golgi traffic. Mol. Biol. Cell 15, 4798-4806.
Panic, B., Perisic, O., Veprintsev, D. B., Williams, R. L. and Munro, S. (2003a). Structural basis for Arl1-dependent targeting of homodimeric GRIP domains to the Golgi apparatus. Mol. Cell 12, 863-874.[CrossRef][Medline]
Panic, B., Whyte, J. R. C. and Munro, S. (2003b). The Arf-like GTPases Arl1p and Arl3p act in a pathway that interacts with vesicle-tethering factors at the Golgi apparatus. Curr. Biol. 13, 405-410.[CrossRef][Medline]
Perez, F., Pernet-Gallay, K., Nizak, C., Goodson, H. V., Kreis, T. E. and Goud, B. (2002). CLIPR-59, a new trans-Golgi/TGN cytoplasmic linker protein belonging to the CLIP-170 family. J. Cell Biol. 156, 631-642.
Pernet-Gallay, K., Antony, C., Johannes, L., Bornens, M., Goud, B. and Rios, R. M. (2002). The overexpression of GMAP-210 blocks anterograde and retrograde transport between the ER and the Golgi apparatus. Traffic 3, 822-832.[CrossRef][Medline]
Presley, J. F., Cole, N. B., Schroer, T. A., Hirschberg, K., Zaal, K. J. M. and Lippincott-Schwartz, J. (1997). ER-to-Golgi transport visualized in living cells. Nature 389, 81-85.[CrossRef][Medline]
Reaves, B. and Banting, G. (1992). Perturbation of the morphology of the trans-Golgi network following brefeldin A treatment: redistribution of a TGN-specific integral membrane protein, TGN38. J. Cell Biol. 116, 85-94.[Abstract]
Rios, R. M. and Bornens, M. (2003). The Golgi apparatus at the cell centre. Curr. Opin. Cell Biol. 15, 60-66.[CrossRef][Medline]
Rios, R. M., Sanchis, A., Tassin, A. M., Fedriani, C. and Bornens, M. (2004). GMAP-210 recruits -tubulin complexes to cis-Golgi membranes and is required for Golgi ribbon formation. Cell 118, 323-335.[CrossRef][Medline]
Rogalski, A. A. and Singer, S. J. (1984). Associations of elements of the Golgi apparatus with microtubules. J. Cell Biol. 99, 1092-1100.[Abstract]
Saint-Pol, A., Yélamos, B., Amessou, M., Mills, I., Dugast, M., Tenza, D., Schu, P., Antony, C., McMahon, H. T., Lamaze, C. et al. (2004). Clathrin adaptor epsinR is required for retrograde sorting on early endosomal membranes. Dev. Cell 6, 532-538.
Sandvig, K. and van Deurs, B. (2002). Transport of protein toxins into cells: pathways used by ricin, cholera toxin and Shiga toxin. FEBS Lett. 529, 49-53.[CrossRef][Medline]
Santini, F., Marks, M. S. and Keen, J. H. (1998). Endocytic clathrin-coated pit formation is independent of receptor internalization signal levels. Mol. Biol. Cell 9, 1177-1194.
Seaman, M. N. (2004). Cargo-selective endosomal sorting for retrieval to the Golgi requires retromer. J. Cell Biol. 165, 111-122.
Setty, S. R. G., Shin, M. E., Yoshino, A., Marks, M. S. and Burd, C. G. (2003). Golgi recruitment of GRIP domain proteins by ARF-like GTPase 1 (Arl1p) is regulated by Arf-like GTPase 3 (Arl3p). Curr. Biol. 13, 401-404.[CrossRef][Medline]
Short, B., Preisinger, C., Schaletzky, J., Kopajtich, R. and Barr, F. A. (2002). The Rab6 GTPase regulates recruitment of the dynactin complex to Golgi membranes. Curr. Biol. 12, 1792-1795.[CrossRef][Medline]
Siniossoglou, S., Peak-Chew, S. Y. and Pelham, H. R. B. (2000). Ric1p and Rgp1p form a complex that catalyses nucleotide exchange on Ypt6p. EMBO J. 19, 4885-4894.
Sprong, H., Degroote, S., Claessens, T., van Drunen, J., Oorschot, V., Westerink, B. H. C., Hirabayashi, Y., Klumperman, J., van der Sluijs, P. and van Meer, G. (2001). Glycosphingolipids are required for sorting melanosomal proteins in the Golgi complex. J. Cell Biol. 155, 369-380.
Storrie, B., White, J., Röttger, S., Stelzer, E. H. K., Suganuma, T. and Nilsson, T. (1998). Recycling of Golgi-resident glycosyltransferases through the ER reveals a novel pathway and provides an explanation for nocodazole-induced Golgi scattering. J. Cell Biol. 143, 1505-1521.
Stüven, E., Porat, A., Shimron, F., Fass, E., Kaloyanova, D., Brügger, B., Wieland, F. T., Elazar, Z. and Helms, J. B. (2003). Intra-Golgi protein transport depends on a cholesterol balance in the lipid membrane. J. Biol. Chem. 278, 53112-53122.
Tai, G., Lu, L., Wang, T. L., Tang, B. L., Goud, B., Johannes, L. and Hong, W. (2004). Participation of the syntaxin 5/Ykt6/GS28/GS15 SNARE complex in transport from the early/recycling endosome to the TGN. Mol. Biol. Cell 15, 4011-4022.
Takahashi, M., Shibata, H., Shimakawa, M., Miyamoto, M., Mukai, H. and Ono, Y. (1999). Characterization of a novel giant scaffolding protein, CG-NAP, that anchors multiple signaling enzymes to centrosome and the Golgi apparatus. J. Biol. Chem. 274, 17267-17274.
Tsukada, M., Will, E. and Gallwitz, D. (1999). Structural and functional analysis of a novel coiled-coil protein involved in Ypt6 GTPase-regulated protein transport in yeast. Mol. Biol. Cell 10, 63-75.
van Meer, G. (2002). Cell biology. The different hues of lipid rafts. Science 296, 855-857.
Wagner, M., Rajasekaran, A. K., Hanzel, D. K., Mayor, S. and Rodriguez-Boulan, E. (1994). Brefeldin A causes structural and functional alterations of the trans-Golgi network of MDCK cells. J. Cell Sci. 107, 933-943.
Walenta, J. H., Didier, A. J., Liu, X. and Kramer, H. (2001). The Golgi-associated Hook3 protein is a member of a novel family of microtubule-binding proteins. J. Cell Biol. 152, 923-934.
Warren, G. and Shorter, J. (2002). Golgi architecture and inheritance. Annu. Rev. Cell Dev. Biol. 18, 379-420.[CrossRef][Medline]
Whiteman, E. L., Chen, J. J. and Birnbaum, M. J. (2003). Platelet-derived growth factor (PDGF) stimulates glucose transport in 3T3-L1 adipocytes overexpressing PDGF receptor by a pathway independent of insulin receptor substrates. Endocrinology 144, 3811-3820.
Wu, M., Lu, L., Hong, W. and Song, H. (2004). Structural basis for recruitment of GRIP domain golgin-245 by small GTPase Arl1. Nat. Struct. Mol. Biol. 11, 86-94.[CrossRef][Medline]
Wu, X., Rao, K., Bowers, M. B., Copeland, N. G., Jenkins, N. A. and Hammer, J. A. (2001). Rab27a enables myosin Va-dependent melanosome capture by recruiting the myosin to the organelle. J. Cell Sci. 114, 1091-1100.
Xu, Y., Takeda, S., Nakata, T., Noda, Y., Tanaka, Y. and Hirokawa, N. (2002). Role of KIFC3 motor protein in Golgi positioning and integration. J. Cell Biol. 158, 293-303.
Yoshino, A., Bieler, B. M., Harper, D. C., Cowan, D. A., Sutterwala, S., Gay, D. M., Cole, N. B., McCaffery, J. M. and Marks, M. S. (2003). A role for GRIP domain proteins and/or their ligands in structure and function of the trans Golgi network. J. Cell Sci. 116, 4441-4454.
Related articles in JCS: