Construction of VSVGtsO45 Fusion Proteins
VSVGtsO45 contains a single amino acid change within its lumenal domain that leads to its misfolding and retention within the ER at 40°, but not at 32°C (Gallione and Rose, 1985
). To study quality control features of the secretory pathway such as retention in and retrieval to the ER, we attached the thermosensitive lumenal domain of VSVGtsO45 to proteins and/or targeting domains that normally direct localization to the Golgi complex or plasma membrane (Fig. ). These included: the KDELR, a Golgi protein that recycles escaped KDEL-containing ligands from the Golgi to the ER (Lewis and Pelham, 1992
); KDELR mutant (KDELRm), a recycling-defective form of KDELR (Townsley et al., 1993
); the transmembrane domain and cytoplasmic tail of TGN38, a protein characteristic of the TGN (Humphrey et al., 1993
); and the transmembrane domain and cytoplasmic tail of the α chain of the IL2R, which functions in IL2 uptake at the cell surface (Leonard et al., 1984
). We also replaced the transmembrane domain of VSVGtsO45 with a synthetic stretch of 15 leucine residues, which previously has been shown to cause Golgi accumulation of proteins normally destined to the plasma membrane (Munro, 1995
Figure 1 Schematic representation of VSVGtsO45 chimeras. The temperature-sensitive lumenal domain of VSVGtsO45 was fused in-frame with (left to right): a full-length human homologue of the KDELR (VSVG–KDELR; Hsu et al., 1992); a full-length mutant (more ...)
Expression and Localization of VSVG Fusion Proteins
The cellular distributions and biochemical modifications of VSVGtsO45 and each of the fusion proteins were characterized in transfected COS cells by indirect immunofluorescence microscopy using polyclonal anti-VSV antiserum. At the nonpermissive temperature of 40°C, each of the chimeras, as well as parental VSVGtsO45, showed a reticular ER-like pattern with nuclear envelope staining (Fig. , left
), which coincided with labeling of endogenous ER membrane proteins (data not shown). Metabolic pulse-chase labeling experiments of transfected cells maintained at 40°C revealed that fusion proteins remained sensitive to endo H digestion (Fig. , right
), suggesting they were not transported out of the ER into the secretory pathway at 40°C where their N
-linked glycans would be modified by Golgi enzymes. Therefore, the VSVGtsO45 lumenal domain appeared to be sufficient for retention of the fusion proteins in the ER at 40°C, consistent with the temperature-induced folding defect demonstrated for VSVGtsO45 (Doms et al., 1987
Figure 2 Localization of VSVGtsO45 chimeras at nonpermissive (40°C) and permissive (32°C) temperatures. COS cells were transiently transfected with the indicated constructs and maintained at either 40° or 32°C. After 40 h, cells (more ...)
Figure 3 Processing of VSVG chimeras at nonpermissive and permissive temperatures. COS cells were pulse labeled with [35S]methionine for 20 min at 40°C, and then chased at 32°C in unlabeled medium for 2 h, or maintained at 40°C for 4 h. (more ...)
Whether the appended VSVGtsO45 ectodomain could properly fold and permit targeting of the chimeras to their predicted destinations was tested in COS cells transfected at 32°C (Fig. , right
). At this temperature, VSVG–KDELR localized predominantly to the Golgi complex with moderate amounts of ER staining. The VSVG–KDELR mutant chimera, by contrast, gave an exclusively Golgi staining pattern when expressed at 32°C, without associated ER staining. Overexpression of KDEL-containing molecules at 32°C induced the redistribution of the KDELR wild-type, but not mutant, chimera into the ER (data not shown). This is consistent with the results of Townsley et al. (1993)
, and suggests that the G protein ectodomain does not interfere with KDELR function (Lewis and Pelham, 1992
). VSVG– TGN38 was similarly targeted to the Golgi complex at 32°C, and could be redistributed to the cell surface by overexpression of other TGN-targeted molecules, similar to results of Humphrey et al. (1993)
for TGN38 (data not shown).
The steady-state Golgi distributions of the above chimeras persisted in cells treated with cycloheximide, which blocks new protein synthesis, indicating they were stably maintained within Golgi membranes. This contrasted with VSVGtsO45, VSVG–IL2R, and VSVG–Leu15, which were delivered to the plasma membrane at 32°C, although with different efficiencies. There was a significant accumulation of the Leu15 (and to a lesser extent IL2R) chimera in the Golgi complex (Fig. , right), but this pool could be completely chased to the plasma membrane with cycloheximide treatment after several hours (data not shown). This suggested that insertion of the Leu15 transmembrane domain slowed transport of VSVG through the Golgi complex, rather than specifically targeting the fusion protein to the Golgi complex (Munro et al., 1995).
In metabolically labeled cells pulsed at 40°, and then chased at 32°C for 2 h, the vast majority of the VSVG– KDELR remained sensitive to endo H (Fig. ). By contrast, ~50% of the VSVG–KDELRm acquired endo H resistance and neuraminidase sensitivity, suggesting this chimera has access to the trans-most processing enzymes of the Golgi complex. VSVG–TGN38 gave a more complex carbohydrate processing pattern with several endo H–resistant bands, including partial sensitivity to neuraminidase. Although the reason for the lack of complete processing of these chimeras is unclear, these results suggest that the KDELRm and TGN38 chimeras become more accessible to distal Golgi processing enzymes than the KDELR wild-type chimera. Both VSVG–Leu15 and VSVG–IL2R became partially endo H resistant and neuraminidase sensitive (Fig. ), and were fully processed upon longer chase periods (data not shown), similar to parental VSVGtsO45.
Laser scanning confocal microscopy was used to compare the distributions of the VSVG chimeras with other well-characterized Golgi markers (see Nilsson et al., 1993
; Shima et al., 1997
). Transfected cells were maintained at 32°C, treated with cycloheximide for 3 h, fixed, and labeled with appropriate primary and secondary antibodies, and then optical sections were analyzed. Fig. shows differences in distributions revealed by overlay images from single optical sections (depth of field is ~0.4 μm). The distribution of each of the chimeras extended throughout the Golgi complex, as evidenced by partial colocalizations with the cis
-Golgi membrane protein GM130 (Nakamura et al., 1995
), the medial Golgi enzyme mannosidase II (Rabouille et al., 1995
), and the TGN marker, furin (Bosshart et al., 1994
). However, clear gradations of distribution existed, even in transiently expressing cells. VSVG–KDELR showed the greatest degree of overlap with GM130 and mannosidase II, although it can be seen to extend into the TGN, whereas VSVG–KDELRm and VSVG–TGN38 were significantly concentrated within the TGN (see overlay with furin), with less accumulation in the proximal regions of the Golgi (GM130 and mannosidase II).
Figure 4 Colocalization of VSVG chimeras with Golgi markers by immunofluorescence microscopy. COS cells transfected at 32°C with the indicated chimeras and indicated Golgi marker proteins (GM130 shows endogenous protein) were treated with cycloheximide (more ...)
Taken together, these results show that addition of even full-length proteins to the ectodomain of VSVGtsO45 permitted not only the proper thermoreversibility of the G protein ectodomain, but the proper targeting specificity dictated by the attached molecules and/or domains.
Retrograde Transport of Golgi-localized VSVG Fusion Proteins
To study whether VSVG fusion proteins ever return to the folding environment of the ER once transported into the Golgi complex, we examined the effect of a temperature shift to 40°C on the distribution of Golgi-localized chimeras. Assuming the ER is the only compartment where misfolding of proteins leads to their retention (Hurtley and Helenius, 1989
), any of the Golgi-localized fusion proteins that recycled to the ER would be predicted to become misfolded and trapped there over time in cells shifted to 40°C.
Cells transfected with VSVGtsO45 or the various chimeras at 40°C were shifted to 32°C for 2 h (for VSVG– KDELR, VSVG–KDELRm, and VSVG–TGN38), or to 18°C for 3 h (for VSVGtsO45, VSVG–IL2R, and VSVG– Leu15) to accumulate these proteins within the Golgi complex (Fig. , left). Cells were then shifted to 40°C for 2 h, fixed, and then examined by immunofluorescence microscopy. As shown in Fig. (middle), whereas VSVGtsO45 and VSVG–IL2R were primarily transported to the plasma membrane after a shift to 40°C, VSVG–KDELR, VSVG– KDELRm, and VSVG–TGN38 redistributed into the ER. VSVG–Leu15 showed an intermediate ER/plasma membrane distribution. Double labeling with antibodies to mannosidase II (Fig. , bottom row), or to the peripheral Golgi coat protein, β-COP (data not shown) showed that redistribution of the fusion proteins into the ER at 40°C did not affect the distribution of other Golgi proteins or disrupt the organization of the Golgi complex. In addition, a wild-type VSVG–TGN38 chimera (whose lumenal domain was not thermosensitive for misfolding) showed no change in its Golgi localization in cells shifted to 40°C (data not shown), indicating the redistributions were specific for fusion proteins containing the tsO45 mutation.
Figure 5 Membrane cycling dynamics of VSVG chimeras. COS cells transfected at 40°C were shifted to either 18°C for 3 h (VSVGtsO45, IL2R, and LEU15 chimeras), or to 32°C for 2 h (KDELR wild type, KDELRm, and TGN 38 chimeras; left), (more ...)
Several observations suggested that the accumulation of the fusion proteins within the ER reflected their retrograde transport into the ER, rather than degradation of the Golgi-localized proteins and ER retention of newly synthesized pools at 40°C. First, redistribution of the fusion proteins into the ER occurred in the absence of new protein synthesis, since transfected cells treated with 50– 100 μg/ml cycloheximide (sufficient to inhibit protein synthesis in these cells by 85–90%) gave the same pattern and kinetics of redistribution as untreated cells (data not shown). Second, there was no evidence for a significant loss of material during the temperature shift as revealed from pulse-chase/immunoprecipitation experiments (see Fig. ). Finally, in biochemical assays with organelles that had been separated by gradient fractionation, the percentage of metabolically labeled VSVG–TGN38 in Golgi membranes decreased in cells shifted from 32° to 40°C for 2 h, with a corresponding increase in ER-enriched fractions (data not shown).
Figure 9 The relationship between misfolding and recycling in intact cells. COS cells expressing VSVG–TGN38 were pulse labeled at 40°C, and then chased in unlabeled medium at 32°C for either 5 min or 2 h. After each chase point, equal (more ...)
Cells expressing the KDELR, KDELRm, and TGN38 chimeras that were shifted to 40°C showed normal Golgi staining patterns when subsequently returned to 32°C (Fig. , right). Thus, the ER accumulation phenotype of these proteins is fully reversible. These results indicate that it is possible to trap and accumulate within the ER at 40°C tsO45-containing proteins that have been recycled from all Golgi compartments.
Kinetics and Properties of Retrograde Transport
To compare the kinetics of redistribution into the ER for the different Golgi resident chimeras, cells maintained at 32°C were shifted to 40°C for various lengths of time, fixed, and then examined by immunofluorescence microscopy. The length of time at 40°C necessary to redistribute each of the chimeras completely back into the ER was then determined and was used to estimate recycling kinetics. VSVG– KDELR redistributed into the ER with a t1/2 of ~20 min, whereas VSVG–TGN38 and VSVG–KDELRm redistributed more slowly (t1/2, ~60 and ~75 min, respectively). Thus, each of the fusion proteins exhibited a distinct rate of transport to the ER, which appeared to be fastest for proteins localized in the cis-most regions of the Golgi and slower for proteins localized in the trans-Golgi/TGN.
Several drug treatments were found to inhibit redistribution of the VSVGtsO45 fusion proteins into the ER upon a temperature shift from 32° to 40°C. These included: ATP depletion with 2-deoxy-glucose/sodium azide; AlF4
, an activator of heterotrimeric G proteins; forskolin, an antagonist of BFA; W7, a calmodulin antagonist; wortmannin, a phosphatidylinositol-specific kinase inhibitor; concanamycin B, an inhibitor of vacuolar proton-ATPases; and the Na+
ionophore, monensin (Table ; refer to Materials and Methods). CHO cells stably expressing VSVG–TGN38 were incubated at 32°C to accumulate the chimera in the Golgi complex, and again for an additional 10 min with the indicated drugs at 32°C before shifting to 40°C for 2 h. Cells were fixed and then examined by immunofluorescence microscopy. All of the above treatments, excluding the ionophore monensin, effectively prevented redistribution of VSVG–TGN38 into the ER at 40°C without changing its Golgi distribution. This was compared with the redistribution of Golgi membranes into the ER by BFA (Doms et al., 1989
; Lippincott-Schwartz et al., 1989
). Interestingly, all of the above treatments, including monensin, also inhibited retrograde transport of VSVG–TGN38 into the ER with BFA (Table ). Although the molecular mechanisms through which these compounds prevent retrograde traffic are unclear, these results suggest that membrane pathway(s) from the Golgi to the ER, followed by the fusion proteins during temperature shifts and BFA treatment, share similar, but not identical, properties.
Inhibition of Retrograde Membrane Traffic
Recycling of Itinerant VSVG Fusion Proteins
Although VSVGtsO45 was delivered exclusively to the cell surface when shifted to 40°C after first being accumulated in the Golgi complex (Fig. , middle), a significant proportion of VSVG–Leu15 and VSVG–IL2R, most noticeably VSVG–Leu15, had indeed redistributed back to the ER. Staining of unfixed nonpermeabilized cells with anti-VSVG antibodies showed reduced levels of surface staining for the chimeras as compared with the parent molecule (data not shown). Quantitation of surface expression by flow cytometry (Fig. ) revealed that, although the percentage of cells expressing detectable levels of surface staining were similar (~7–9%), VSVG–Leu15 exhibited mean fluorescence intensity levels significantly lower than VSVGtsO45 (60% of tsO45). VSVG–IL2R showed an intermediate level of cell surface expression (75% of tsO45). Since total expression levels were equivalent when cells were stained in the presence of detergent (data not shown), this result indicates that significantly less VSVG– Leu15 (and VSVG–IL2R) had been delivered to the plasma membrane than parental VSVGtsO45. The remainder (~40% of VSVG–Leu15 and ~25% of VSVG–IL2R) had presumably redistributed to the ER (Fig , middle), since Golgi staining was evident for the Leu15 (and IL2R) chimeras, but not the parent G protein when the temperature was subsequently lowered from 40° to 32°C (Fig. , right). As these experiments were performed in the presence of cycloheximide, this material must have originated from previously redistributed Golgi pools.
Figure 6 Surface staining of VSVG chimeras by flow cytometry. COS cells were transiently transfected with 5 μg VSVGtsO45, or the LEU15, IL2R, or KDELR wild-type chimeric plasmids, and then maintained at 40°C. At 40 h posttransfection, cycloheximide (more ...)
The observation that VSVGtsO45, –IL2R, and –Leu15 were delivered from the ER to the Golgi complex upon a shift from 40° to 32°C (or 18°C) at approximately equivalent rates (data not shown; Fig. , left) implies that it is their transport through the Golgi complex, rather than exit from the ER, that is slowed when compared with VSVGtsO45. When combined with the recycling and biochemical processing data, these results suggest that whereas proteins rapidly transported through the Golgi complex (VSVGtsO45) continue to the cell surface after the temperature shift, proteins that are either stably retained (VSVG–KDELR, –KDELRm, and –TGN38) or slowly transported through the Golgi (VSVG–Leu15 and IL2R) have the capacity to be retrieved to the ER.
Folding Properties of VSVG Fusion Proteins in the ER and Golgi Complex
The redistribution of VSVG chimeras into the ER after a temperature shift to 40°C is presumed to be due to misfolding/retention of the G protein ectodomain within the ER as these molecules recycle from the Golgi complex. This interpretation was tested using the conformation-specific VSVG antibody, I14 (Lefrancois and Lyles; 1982), which has previously been used to detect folded, but not misfolded, VSVG molecules (Doms et al., 1988
; Machamer et al., 1990
). As shown in Fig. (a
), I14 staining of VSVG–TGN38 in stably expressing CHO cells gave a primarily Golgi staining pattern at 32°C. As in COS cells, this staining coincided with β-COP (data not shown) and the Golgi enzyme mannosidase II (Fig. b
). After short periods at 40°C, before VSVG–TGN38 had redistributed into the ER (data not shown), or under conditions where its redistribution was inhibited by ATP depletion (Fig. e
), VSVG–TGN38 staining by I14 antibodies was still detected in the Golgi complex. This indicated that VSVG– TGN38 did not significantly misfold when it was in the Golgi complex at 40°C, even after several hours during which recycling was inhibited (e.g., with ATP depletion).
Figure 7 Analysis of cycling using conformation-sensitive antibodies. CHO cells stably expressing VSVG–TGN38 were treated as indicated and stained with conformation-sensitive I14 antibodies to VSVG (a, c, e, g, i, and k), and antibodies to the Golgi enzyme, (more ...)
Under conditions where VSVG–TGN38 was transported into the ER (i.e., upon longer periods at 40°C), specific staining for VSVG–TGN38 by I14 was lost (Fig. c
), but reappeared when cells were shifted from 40° back to 32°C for 10 min under conditions where export from the ER was blocked (e.g., ATP depletion; Fig. g
). Loss of I14 staining of VSVG–TGN38 in the ER at 40°, but not at 32°C, was also observed in cells with Golgi proteins redistributed into the ER with BFA (Doms et al., 1989
; Lippincott-Schwartz et al., 1989
; Fig. , compare i
). These results are consistent with the idea that the mechanism for the accumulation of the VSVG chimeras within the ER in cells transfected at 40°C and during the 40°C temperature shift is due to misfolding of the G protein ectodomain in this compartment. Moreover, changes in conformation of VSVG–TGN38 leading to loss of labeling by I14 antibodies appeared to occur only after the protein had redistributed into the ER.
Biochemical analysis provided further evidence that misfolding of the chimeras at 40°C occurred only after the proteins had redistributed into the ER. A cell fractionation procedure to separate ER from Golgi membranes was developed to analyze misfolding in vitro. COS cells expressing VSVG–TGN38 were metabolically labeled with [35S]methionine at the permissive temperature to generate labeled proteins in both the ER and Golgi complex. Cells were gently scraped, pelleted, and then a postnuclear supernatant was layered over a continuous 0–26% OptiPrep gradient (refer to Materials and Methods). Fractions were collected and analyzed for the distribution of ER (ribophorin) and Golgi (galactosyltransferase) membrane markers (Fig. a). Separate ER and Golgi fractions were pooled and aliquots were incubated at either 32° or 40°C for 60 min to assess the capacity of the individual membrane compartments to misfold VSVG–TGN38. After solubilization at the indicated temperatures, VSVG–TGN38 was immunoprecipitated with either polyclonal antibodies that recognize both folded and misfolded G protein, or with the conformation-sensitive I14 antibody.
Figure 8 Fractionation of ER and Golgi membranes and misfolding in vitro. (A) COS cells transfected with VSVG–TGN38 were labeled with [35S]methionine, chased to generate labeled molecules in both the ER and Golgi complex, and then homogenized and (more ...)
As shown in Fig. (b
), polyclonal anti-VSV recognized VSVG–TGN38 in both ER and Golgi fractions regardless of the incubation temperature. I14 was also able to precipitate VSVG–TGN38 from fractions containing Golgi and ER membranes at 32°C. However, incubation at 40°C dramatically reduced the ability of I14 to recognize VSVG– TGN38 from ER fractions, whereas immunoprecipitation from Golgi fractions was only slightly affected. Therefore, consistent with de Silva et al. (1990)
, only the ER appears to contain specific factors capable of misfolding tsO45-containing chimeras at the nonpermissive temperature.
We next determined whether we could observe the recycling/misfolding process in intact cells biochemically. COS cells expressing VSVG–TGN38 were metabolically labeled at 40°C, chased in unlabeled medium at 32°C for either 5 min, where the majority of newly synthesized molecules should still be in the ER, or for 2 h to accumulate the labeled chimera in the Golgi complex, before shifting back to 40°C for an additional period of time. After solubilization at the indicated temperatures, VSVG–TGN38 was immunoprecipitated with either polyclonal or conformation-sensitive I14 antibodies. As shown in Fig. , whereas VSVG–TGN38 could fold completely after a 5-min chase at 32°C, only 25% could be immunoprecipitated with I14 after a subsequent shift to 40°C. Thus, folded VSVG– TGN38 can rapidly misfold when it is contained within the environment of the ER. However, when VSVG–TGN38 had been chased for 2 h at 32°C and then shifted to 40°C, >80% remained properly folded after a 10-min incubation at 40°C. It is likely that the remaining misfolded fraction represents a population of VSVG–TGN38 molecules that has not left the ER. Therefore, as with the parent G protein (de Silva et al., 1990
), and consistent with the in vitro data (Fig. ), the chimera becomes resistant to misfolding when it is delivered to the Golgi complex. If, after a 2-h chase at 32°C, labeled cells were shifted to 40°C for increasing periods of time, the amount of I14 precipitable material decreased, whereas material recognized by polyclonal anti-VSV remained unchanged (Fig. ). The temporal loss of I14 immunoreactivity, therefore, correlated with recycling and subsequent misfolding of VSVG–TGN38 as it redistributed into the ER. This misfolding could be reversed by a subsequent short incubation at 32°C (Fig. ). Thus, the fusion proteins do not appear to misfold in the Golgi complex upon a shift to 40°C, but only once they have recycled into the ER.