Effects of Dominant Mutants of Arfs on Golgi Morphology and Coat Protein Recruitment
BFA and dominant mutant forms of Arf1 have been used to demonstrate important roles for Arf1 in the morphology of the Golgi apparatus and recruitment of coat proteins to the Golgi. BFA causes the rapid (<2 min at 5 μg/ml) loss of coat proteins, such as COPI (viewed with an antibody to the β-subunit, βCOP), from Golgi membranes to the cytosol and causes the slower (>10-min) dissolution of the Golgi in intact cells. Similarly, dominant negative forms of Arf1 exhibit a BFA-like phenotype in which the Golgi collapses and coat proteins bound to the Golgi become soluble. Conversely, the dominant active [Q71L]Arf1 causes the vesiculation and expansion of the Golgi and increased stability of COPI association with Golgi membranes (Zhang et al., 1994
). To determine whether other Arfs perform similar roles in Golgi morphology and coat protein recruitment, we created NRK cells stably transfected with wild type, dominant active, and dominant negative forms of human Arf3 and Arf4 under control of the interferon-inducible Mx1 promoter. We were able to generate stable cell lines capable of inducible expression of Arf5, but not either of the Arf5 mutants. The reason for our failure to obtain mutant Arf5-expressing clones is unknown but may result from leakiness (although no increases in any Arf proteins have been observed by immunoblotting in the absence of induction) of the Mx1 promoter and greater sensitivity of NRK cells to Arf5 mutants than the mutants of other Arf isoforms.
Interferon induction increased expression of wild type and mutant forms of human Arf3 and Arf4 over endogenous protein () with very similar kinetics to each other and to Arf1 constructs (Zhang et al., 1994
). The induced proteins reached maximal levels of induction within 8–12 h and were stable for at least an additional 24 h (). Although changes in the levels of the dominant active mutants were subtler, the appearance of altered Golgi morphologies correlated temporally with protein induction.
Figure 1. [Q71L]Arf3 expression caused an expansion of the Golgi and delayed the BFA-induced release of βCOP, whereas [N126I]Arf3 caused a dispersion of the Golgi and dissociation of βCOP. (A) NRK cells stably transfected with wild-type and mutant (more ...)
Immunofluorescent staining of the lumenal Golgi protein mannosidase II showed that induction of dominant active [Q71L]Arf3 caused an expansion of the Golgi apparatus (). Induction of wild-type Arf3 did not produce any detectable changes in Golgi morphology. Expression of [Q71L]Arf3 also caused a delayed release of βCOP from the Golgi in response to BFA (). In contrast, expression of dominant negative [N126I]Arf3 caused the Golgi to show a dispersed localization throughout the cell () and caused βCOP to redistribute from the Golgi to the cytosol (). Induction of dominant active and dominant negative Arf4 produced changes in Golgi and βCOP distribution similar to mutant forms of Arf3 (data not shown). However, at any time point, only a smaller percentage of cells was affected. The changes in Golgi morphology and sensitivity to BFA produced by [Q71L]Arf3 and [N126I]Arf3 were similar to those described previously for dominant mutant forms of Arf1 (Zhang et al., 1994
). Therefore, BFA, dominant active and dominant negative forms of Arf1, Arf3, and Arf4 produced similar effects on Golgi morphology and βCOP localization. These data suggest that there were no isoform-dependent effects of dominant mutants of Arfs1–4 on Golgi morphology or coat protein recruitment.
Decreasing Expression of Individual Arfs Produced Isoform-specific Effects on Golgi Morphology
Because at least some Arf GAPs and Arf GEFs can act on multiple Arfs (for reviews, see Donaldson and Jackson, 2000
; Jackson and Casanova, 2000
), the inactivation of these enzymes by one Arf isoform can lead to indirect changes in the level of activation of other Arf subtypes. Thus, neither BFA nor the dominant mutants of Arfs can be assured of selectively altering activity of a single Arf isoform.
We used siRNAs expressed by pSUPER-based plasmids to specifically decrease expression of Arf1, Arf3, Arf4, or Arf5 individually or in paired combinations. For each Arf isoform four different sequences, each 19 nt in length, were chosen from the coding regions. Only plasmids that provided >60% knockdowns of the individually targeted Arf were used. This resulted in two different plasmids that expressed siRNAs targeted to different regions of the Arf sequence (Arf1a, Arf1b, Arf3a, Arf3b, Arf4a, Arf4b, Arf5a, Arf5b). Having two independent plasmids for each Arf isoform thus allowed us to confirm that the phenotypes observed were produced by decreased expression of the respective Arf and not a result of off-target effects produced by expression of an individual siRNA. Expression of siRNAs maximally decreased expression of each Arf by 3 d after transfection and expression remained reduced 4 d after transfection.
Each siRNA plasmid was transfected into HeLa cells, and total cell lysates were analyzed by immunoblotting to determine the effectiveness of each siRNA plasmid. Expression levels were decreased as follows: Arf1a, 92%; Arf1b, 90%; Arf3a, 79%; Arf3b, 75%; Arf4a, 68%; Arf4b, 75%; Arf5a, 77%; and Arf5b, 70% (). Knockdown of each Arf was specific in that only the targeted Arf was decreased in expression () as determined by quantitative immunoblotting. Therefore, siRNAs allowed specific knockdown of endogenous Arf expression, providing a powerful tool for analyzing the functions of individual Arfs.
Figure 2. Knockdown of each Arf isoform by siRNA is effective and specific. (A) HeLa cells were transfected with pSUPER-based plasmids directing expression of siRNAs targeted to human Arf1, Arf3, Arf4, or Arf5 or the empty pSUPERvector. Two different plasmids expressing (more ...)
Decreasing Arf activity with BFA or dominant negative Arf mutants causes substantial changes to the morphology of the various organelles and association of coat proteins with membranes. We found, however, that the loss of any one Arf did not produce a similar result. Decreased expression of Arf1, Arf3, Arf4, or Arf5 did not produce any obvious effects on the morphology of the Golgi apparatus or early endosomes as visualized by immunofluorescent staining for giantin or the transferrin receptor, respectively. In addition, individual Arf knockdowns did not noticeably affect localization of the coat proteins COPI, visualized by βCOP staining, or AP-1, visualized by γ-adaptin staining (our unpublished data). These data were interpreted to suggest either that there exist “spare” Arfs and that up to 92% depletion of a single Arf was still not sufficient to make the cell functionally null for that protein or that there is a level of functional redundancy within the four soluble human Arfs. Note also that the extent of knockdown achieved is limited by the transfection frequency of HeLa cells. Thus, with 92% depletion in the entire cell population and <100% transfection efficiency, we expect many cells to be essentially 100% depleted for ARF1 and thus the lack of change in COPI or AP-1 staining can be taken as strong evidence for redundancy in the roles of ARF1 in recruitment of these coats.
To test the hypothesis that functional redundancy exists among the four soluble Arfs in intact cells, we asked whether knocking down two Arf isoforms results in phenotypes that might be reminiscent of BFA or negative dominant mutant expression. Coexpression of each pair of Arfs substantially decreased expression of the respective isoforms ().
Effects on the cis/medial-Golgi
Decreasing cellular Arf activity with BFA or dominant negative Arf mutants causes dramatic changes in Golgi morphology and functions (Fujiwara et al., 1988
; Lippincott-Schwartz et al., 1989
). Therefore, we asked whether the double knockdowns produced a similar result. In untreated, control (empty pSUPER vector)-transfected cells, the Golgi exhibited a perinuclear localization, as visualized by immunofluorescent staining of the cis
/medial marker GM130 (). Decreased expression of Arf1+Arf4 caused a dramatic change in Golgi morphology; it seemed more punctate with elongated tubules extending from the perinuclear region (, top third and fourth from the left). Quantitation revealed that ~75% of Arf1+Arf4 knockdown cells showed a punctate/tubulated Golgi (), whereas only ~5% of cells in the vector-transfected control cells and <20% of every other pairwise double siRNA combinations showed such a staining pattern for the Golgi. Note that because the transfection efficiency of our HeLa cells is <100%, and we do not correct for this in scoring phenotypes, the actual percentages of doubly transfected cells showing any of the phenotypes we quantify is actually greater than the numbers indicated. None of the other paired combinations of Arf knockdowns caused a perceptible change in GM130 staining. BFA treatment caused GM130 to localize to puncta distributed throughout the cytoplasm (, bottom, fourth from left) (Nakamura et al., 1995
), whereas giantin seemed completely dispersed throughout the cytoplasm (, third from left) (Linstedt and Hauri, 1993
). Similar to the effects of BFA, decreased expression of Arf1+Arf4 caused giantin to seem dispersed (, second from left). The specificity of these changes to the combination of Arf1 and Arf4 were confirmed with independent sets of plasmids; i.e., Arf1a+Arf4b and Arf1b+Arf4a produced the same effects. These data suggest that the effects of BFA on Golgi morphology result from inhibition of Arf1 and Arf4, although changes resulting from depletion of Arf1+Arf4 are not identical to those from BFA treatment.
Effects of Decreased Arf Expression on COPI Localization
In addition to effects on Golgi morphology, Arfs recruit COPI to the Golgi. βCOP is a component of the COPI coatomer complex that mediates traffic between the Golgi cisternae, from the intermediate compartment to the cis
-Golgi, and from the cis
-Golgi back toward the ER. Dominant negative Arf1 (Zhang et al., 1994
), dominant negative Arf3 (), or BFA () caused a redistribution of βCOP from the Golgi to the cytosol. Impaired COPI recruitment may contribute to the changes in Golgi morphology because coats have been proposed to induce membrane curvature and mediate budding of vesicles. We thus tested the effects of decreased expression of Arf isoforms on recruitment of COPI to the Golgi.
Figure 6. Reduced levels of Arf1+Arf3 and Arf1+Arf4 trap ts045-VSVG-GFP early in the secretory pathway. HeLa cells were cotransfected with pSUPER-based and ts045-VSVG-GFP plasmids and incubated as described in the legend to . (A) Cells were then incubated (more ...)
In pSUPER-transfected control cells and the Arf3+Arf4, Arf3+Arf5 and Arf4+Arf5 double knockdowns, βCOP showed perinuclear localization, consistent with its unchanged localization to the Golgi and to small puncta throughout the cytoplasm (). In contrast, decreased expression of Arf1+Arf4 caused βCOP to seem dispersed throughout the cytoplasm (, top, third from left), similar to the effects of BFA treatment (, bottom, fourth from left). These data were confirmed with independent sets of plasmids, i.e., Arf1a+Arf4b and Arf1b+Arf4a produced the same effects. Cell counts revealed that ~58% of Arf1+Arf4 knockdown cells showed dispersed βCOP localization (), whereas <5% of cells in the vector-transfected control cells or <20% of any of the other combinations of double siRNA combinations showed such a staining pattern for the βCOP.
Figure 4. Decreased expression of Arf1+Arf4 caused βCOP to disperse throughout the cytoplasm, whereas Arf1+Arf3 and Arf1+Arf5 double knockdowns caused βCOP to localize to large cytoplasmic puncta. (A) After 3 d, control pSUPER- and siRNA-transfected (more ...)
In Arf1+Arf3 and Arf1+Arf5 knockdown cells, the peripheral cytoplasmic puncta to which βCOP localized seemed larger compared with pSUPER-transfected control cells. We identified these puncta as belonging to the ER-Golgi intermediate compartment (ERGIC, aka vesicular tubular clusters, VTCs; see below and Figures and ). Quantitation demonstrated that ~73% of Arf1+Arf3 and ~54% of Arf1+Arf5 knockdown cells showed βCOP localization to enlarged, cytoplasmic puncta (), whereas <10% of cells in the vector-transfected control cells or the other double siRNA combinations showed such a staining pattern for βCOP. It is notable that accumulation of βCOP in cytoplasmic puncta in the Arf1+Arf3 and Arf1+Arf5 knockdowns was more apparent in the ts045-VSVG-GFP experiments (see below), indicating that a bolus of protein transiting the secretory pathway exacerbates the effects of Arf depletions on βCOP localization in these cells. Therefore, decreased expression of both Arf1 and Arf4 caused βCOP to disperse throughout the cytosol, similar to the effects of BFA, whereas Arf1+Arf3 and Arf1+Arf5 knockdowns cause accumulation of βCOP in enlarged VTCs. Together, these data indicate that Arf1 plays a central role in traffic through the Golgi but that each of the other cytoplasmic Arfs (Arf3–5) makes specific contributions to the cellular functions of Arfs at different steps in the early secretory pathway.
Effects of Decreasing Arf Expression on Anterograde Traffic through the Secretory Pathway
Dominant mutants of Arf1 or BFA treatment inhibit traffic of secreted proteins, such as the vesicular stomatitis G protein (VSVG), through the secretory pathway (Doms et al., 1989
; Dascher and Balch, 1994
; Zhang et al., 1994
; Aridor et al., 1995
; Rowe et al., 1996
). However, other treatments that alter Golgi morphology, such as dispersion of the Golgi by microtubule depolymerization, do not impair secretory traffic (Cole et al., 1996
). We therefore used the temperature-sensitive mutant of ts045-VSVG, tagged with green fluorescent protein (ts045-VSVG-GFP), to determine altered Golgi structure and COPI localization produced by the Arf double knockdowns correlated with altered protein traffic through the early secretory pathway. At 40°C, ts045-VSVG-GFP is misfolded and retained in the ER (Scales et al., 1997
). After switching to the permissive temperature for ts045-VSVG-GFP folding (1.5 h at 32°C), ts045-VSVG-GFP in control cells was no longer in the ER but rather was seen at the plasma membrane, where it colocalized with the cell surface marker ConA, and a perinuclear location, consistent with Golgi (, top, Hirschberg et al., 2000
). No differences from controls were found in the Arf1+Arf5, Arf3+Arf4, Arf3+Arf5, and Arf4+Arf5 cells. However, in Arf1+Arf4 knockdown cells, ts045-VSVG-GFP did not travel to the plasma membrane and did not colocalize with ConA (, third row from top). Instead, after shifting to the permissive temperature, ts045-VSVG-GFP in the Arf1+Arf4 knockdowns remained in the ER, where it colocalized with the ER marker calnexin (), similar to effects of BFA treatment and expression of dominant negative Arf1 on ts045-VSVG-GFP traffic. These data were confirmed with independent sets of plasmids, i.e., Arf1a+Arf4b and Arf1b+Arf4a produced the same effects. Quantitation revealed that ~58% of Arf1+Arf4 knockdown cells showed ER localization of ts045-VSVG-GFP after 90 min at the permissive temperature ().
The exit of proteins from the ER is mediated by Sar1 (a distant member of the Arf family) and COPII at ER exit sites (Barlowe et al., 1994
; Kuge et al., 1994
) and ER-derived vesicles exchange COPII for COPI in an Arf-dependent manner and subsequently differentiate into VTCs that then travel to the Golgi (Aridor et al., 1995
; Rowe et al., 1996
; Scales et al., 1997
). We therefore performed double-labeling immunofluorescence experiments to determine at which step in the secretory pathway ts045-VSVG-GFP traffic is inhibited by decreased expression of Arf1+Arf4. Confocal images demonstrate that 90 min after shifting from 40°C to the permissive temperature of 32°C, the ts045-VSVG-GFP colocalized with the ER marker calnexin in cells in which Arf1 and Arf4 expression were reduced (, second row from top). In some cells, ts045-VSVG-GFP also colocalized with ER-GIC53, a marker for VTCs, which mediates traffic from the ER to the Golgi (, fourth row from top). Therefore, Arf1+Arf4 knockdowns inhibited transport of ts045-VSVG-GFP at a very early step in transport from the ER.
In Arf1+Arf3 double knockdown cells, ts045-VSVG-GFP localized to the perinuclear compartment. However, unlike control cells, the perinuclear ts045-VSVG-GFP looked like dramatically enlarged puncta (). Although ts045-VSVG-GFP trafficked to the plasma membrane in Arf1+Arf3 knockdown cells (, second row, panel 1, see arrows), the amount of ts045-VSVG-GFP at the cell surface seemed diminished. These data were confirmed with independent sets of plasmids, i.e., Arf1a+Arf3b and Arf1b+Arf3a produced the same effects. Therefore, decreased expression of Arf1+Arf4 inhibited transport of ts045-VSVG-GFP at an early step in transport from the ER, whereas decreased levels of Arf1+Arf3 caused accumulation of ts045-VSVG-GFP in large perinuclear puncta.
In cells in which Arf1+Arf3 were depleted, the localization of ERGIC53 and thus the morphology of the ER-Golgi intermediate compartment/VTCs was dramatically altered. In control cells, ERGIC53 localized to small puncta localized throughout the cell (, top row). In Arf1+Arf3 knockdowns, ERGIC53 localized to large puncta (, second row), similar to those formed after BFA treatment (Lippincott-Schwartz et al., 1990
). These large ERGIC53-positive puncta colocalized with ts045-VSVG-GFP and βCOP. Although decreased expression of Arf1+Arf5 also resulted in the formation of enlarged βCOP-positive puncta, relative to control cells (), the pattern of ERGIC53 staining seemed normal and ts045-VSVG-GFP did not colocalize in these cells. Quantitation of costaining in confocal images revealed that 42% of Arf1+Arf3 knockdown cells showed ERGIC53 localization to large cytoplasmic puncta (), whereas <5% of cells in the vector-transfected control cells or the other double siRNA combinations showed such a staining pattern for ERGIC53. Therefore, decreased expression of Arf1+Arf3 selectively impaired traffic of βCOP-positive VTC's to the cis
Effects of Decreased Arf Expression on Retrograde Traffic from Golgi to ER
Arfs also play a role in COPI-mediated retrograde traffic from the cis-Golgi back to the ER. We therefore determined the effects of the double knockdowns on the localization of the KDEL receptor, which retrieves proteins containing the KDEL sequence from the cis-Golgi and returns them to the ER. In pSUPER-transfected control cells, the KDEL receptor localized to small puncta that are more concentrated near the nucleus (, far left). Localization of the KDEL receptor seemed similar to control cells in Arf1+Arf4 and Arf3+Arf5 double knockdowns. In Arf3+Arf4 and even more so in Arf4+Arf5 cells, there is a dramatically increased concentration of the KDEL receptor at the cis-Golgi (, third and fifth from the left). Approximately 46 and 64% of Arf3+Arf4 and Arf4+Arf5 double knockdown cells, respectively, show enhanced Golgi localization of the KDEL receptor (, bottom). Because the KDEL receptor normally cycles between the ER and cis-Golgi this change in steady-state distribution likely reflects a decrease in the rate of exit from the cis-Golgi back to the ER.
In 25% of Arf1+Arf3 knockdowns and 36% of Arf1+Arf5 knockdowns, the KDEL receptor seemed less concentrated at the perinuclear region and localized to enlarged puncta in the cell periphery. This finding is similar to the effects of BFA on KDEL receptor localization, which has been shown to arrest the KDEL receptor and other rapidly recycling proteins in the ERGIC (Hauri et al., 2000
). These KDEL receptor-positive puncta colocalized with βCOP-positive puncta (), suggesting a blockage in ERGIC/VTC to cis
-Golgi traffic. But because COPI and the KDEL receptor are each involved in traffic in both directions between the ER and Golgi, we cannot confidently assign directionality to the defects in traffic observed. Overall, these data are interpreted as showing that decreased expression of Arf3+Arf4 or Arf4+Arf5 retards or arrests the KDEL receptor in the cis
-Golgi, whereas in Arf1+Arf3 or Arf1+Arf5 double knockdowns, the KDEL receptor is trapped in the ERGIC/VTCs.
Effects of Decreased Arf Expression on Endosome-to-Plasma Membrane Traffic
To analyze Tfn uptake, cells were continuously treated with Tfn Alexa 488 for 60 min, before cells were washed, fixed, and analyzed. To measure Tfn recycling cells were incubated with Tfn Alexa 488 for 60 min, and then rinsed and incubated in media alone for an additional 60 min. The amount of intracellular Tfn was measured using confocal images and quantitation of the average pixel intensity within the cells (see Materials and Methods). Recycling was calculated as the average pixel intensity after washout, subtracted from the average pixel intensity after 60 min uptake, normalized to the average amount of recycling in the pSUPER-transfected control cells. The double Arf knockdowns did not affect the extent of Tfn endocytosis (, left bar graph). In contrast, every combination except Arf1+Arf4 (Arf1+Arf3, Arf1+Arf5, Arf3+Arf4, Arf3+Arf5, and Arf4+Arf5) significantly impaired Tfn recycling (, right bar graph). The effect was most pronounced with Arf1+Arf3 (~50%) and Arf4+Arf5 (~45%). In the Arf1+Arf3 knockdowns, the endosomes labeled by Tfn Alexa-488 seemed extensively tubulated and after 60 min of Tfn washout, the increased retention of Tfn was evident within these tubules (, middle). In contrast to Arf1+Arf3 double knockdowns, decreased expression of Arf4+Arf5 also increased the retention of Tfn, but in this case, it was trapped in the perinuclear recycling endosomes. The differences in the sites at which Tfn is retained may indicate evidence for distinct roles and sites of action for Arfs in receptor recycling.
Figure 8. Double Arf knockdowns alter the recycling of Tfn. (A) Cells were incubated with Tfn-Alexa488 for 60 min and either fixed or rinsed several times and incubated in media for another 60 min to allow internalized Tfn to return to the cell surface and be released (more ...)