Cell-free transport to distinct Golgi cisternae is compartment specific and ARF independent - PubMed (original) (raw)
Cell-free transport to distinct Golgi cisternae is compartment specific and ARF independent
S Happe et al. J Cell Biol. 1998.
Abstract
The small GTPase ADP-ribosylation factor (ARF) is absolutely required for coatomer vesicle formation on Golgi membranes but not for anterograde transport to the medial-Golgi in a mammalian in vitro transport system. This might indicate that the in vivo mechanism of intra-Golgi transport is not faithfully reproduced in vitro, or that intra-Golgi transport occurs by a nonvesicular mechanism. As one approach to distinguishing between these possibilities, we have characterized two additional cell-free systems that reconstitute transport to the trans-Golgi (trans assay) and trans-Golgi network (TGN assay). Like in vitro transport to the medial-Golgi (medial assay), transport to the trans-Golgi and TGN requires cytosol, ATP, and N-ethylmaleimide-sensitive fusion protein (NSF). However, each assay has its own distinct characteristics of transport. The kinetics of transport to late compartments are slower, and less cytosol is needed for guanosine-5'-O-(3-thiotriphosphate) (GTPgammaS) to inhibit transport, suggesting that each assay reconstitutes a distinct transport event. Depletion of ARF from cytosol abolishes vesicle formation and inhibition by GTPgammaS, but transport in all assays is otherwise unaffected. Purified recombinant myristoylated ARF1 restores inhibition by GTPgammaS, indicating that the GTP-sensitive component in all assays is ARF. We also show that asymmetry in donor and acceptor membrane properties in the medial assay is a unique feature of this assay that is unrelated to the production of vesicles. These findings demonstrate that characteristics specific to transport between different Golgi compartments are reconstituted in the cell-free system and that vesicle formation is not required for in vitro transport at any level of the stack.
Figures
Figure 1
Transport to late Golgi compartments is inhibited by GTPγS at low cytosol concentrations. Inhibition of transport by GTPγS in the medial (A), trans (B), and TGN (C) assays was assessed by titrating CHO cytosol into each assay in the presence of 2 μM GTPγS (closed symbols) or in its absence (open symbols). Data are representative of more than 10 individual experiments in each assay with different cytosol preparations.
Figure 2
ARF can be removed from cytosol in one ion-exchange chromatography step. (A) Bovine brain cytosol was fractionated by chromatography on Fast-Flow Q, as described in Materials and Methods. Closed circles indicate the protein elution profile. The dashed line represents the salt concentration of each elution buffer. Additional buffer components are indicated below the protein peaks. (B) Western blot of unfractionated cytosol, the ARF-containing pool, and the ARF-depleted cytosol probed with anti-ARF monoclonal 1D9 antibody, as described in Materials and Methods. Volume equivalents applied to the gel are indicated below each sample. Data are representative of four independent ARF-depleted cytosol preparations. (C) Western blot of isolated donor and acceptor Golgi membranes (0.5 mg/ml) and unfractionated cytosol, probed with anti-ARF monoclonal 1D9. The blot was overexposed to reveal a slight signal at highest level of acceptor Golgi. No signal was observed with the different donor membranes used in each assay. Only the medial donor Golgi is shown.
Figure 3
ARF-depleted cytosol fully supports transport in all three in vitro assays. Unfractionated bovine brain cytosol (open circles), reconstituted cytosol (closed circles), and ARF-depleted cytosol (open squares) were titrated into the medial (A), trans (B), and TGN (C) assays. A cytosol equivalent is the volume of sample equal to 1 μl of unfractionated control cytosol after taking into account volume changes after chromatography. The protein concentration of control cytosol was 12.1 mg/ml. Data are representative of two independent ARF- depleted cytosol preparations.
Figure 4
ARF depletion prevents cytosol-dependent inhibition by GTPγS. The ability of GTPγS to inhibit transport with bovine brain reconstituted cytosol (circles) or ARF-depleted cytosol (squares) was determined by titrating increasing cytosol equivalents into the medial (A), trans (B), or TGN (C) assays in the presence of 2 μM GTPγS (closed symbols) or in its absence (open symbols). Data are representative of two independent ARF-depleted cytosol preparations.
Figure 5
Recombinant ARF1 restores GTPγS sensitivity to ARF-depleted cytosol. Unfractionated bovine brain cytosol (5 μl) and ARF-depleted cytosol (5-μl equivalents) were tested in the medial (A), trans (B), and TGN (C) assays in the presence of 2 μM GTPγS (solid bars) or in its absence (open bars). The indicated amounts of myr-rARF1 (5.7% myristoylated) or non–myr-rARF1 (hatched bar) were titrated into the assay. Data are a combination of two independent experiments. The maximum counts per minute for unfractionated cytosol were 7,764 ± 184 in the medial assay, 4,076 ± 64 and 2,960 ± 203 in the trans assay, 3,030 ± 204 and 3,389 ± 48 in the TGN assay. The maximum counts per minute for ARF-depleted cytosol were 4,866 ± 114 and 8,850 ± 15 in the medial assay, 2,184 ± 323 and 1,881 ± 15 in the trans assay, and 2,981 ± 153 and 2,498 ± 31 in the TGN assay. The acceptor membranes used in these experiments were different from the preparation used in Fig. 4 and exhibit less cytosol-independent inhibition of transport to the TGN by GTPγS (compare Figs. 4_C_ and 5_C_).
Figure 6
Golgi coated bud formation is blocked by ARF depletion and restored by purified ARF. Replicas of Golgi membranes after a 15-min transport incubation were prepared as described in Materials and Methods. The transport reaction mixtures contained (A) unfractionated bovine brain cytosol (2.4 mg/ml); (B) ARF-depleted bovine brain cytosol (2.4 mg/ml); (C) ARF- depleted cytosol plus myr-rARF1 (120 μg/ml, 5.7% myristorylated); and (D) ARF-depleted cytosol plus non–myr-rARF1 (120 μg/ml). Boxed areas are presented at higher magnification in the panels on the right side of the figure, illustrating the punctate surface coating on Golgi buds in A and C. Buds in B lack this punctate coating and have a granular texture similar to the surrounding tubules. Bars, 0.5 μm.
Figure 7
Depletion of ARF decreases the density of coated buds and vesicles and increases the proportion of cisternae with large fenestrae. (A) The densities of buds and vesicles with a smooth surface (uncoated) or a distinctive punctate coating (coated) were determined from electron micrographs as described in Materials and Methods. Blunt-ended tubules made up greater than 85% of the observed uncoated buds and vesicles in all images. Error bars represent the standard error of the mean, and the numbers in parentheses indicate the number of micrographs analyzed. (B) The average size of cisternal fenestrae was estimated from the electron micrographs. Numbers in parentheses above the bars represent the total number of cisternae. Direct measurement of the maximum diameter of the fenestrae on five micrographs representative of each size class revealed that fenestrae scored as small, medium, and large had average diameters (±SD) of 42 ± 25 nm (n = 32), 84 ± 31 nm (n = 36), and 180 ± 80 nm (n = 33), respectively.
Figure 8
Depletion of ARF does not change the rate of transport. The rate of transport (independent of glycosylation) was assessed in the medial (A), trans (B), and TGN (C) assays in the presence of ARF (unfractionated cytosol, open circles) or the absence of ARF (closed circles) in two-stage assays, as described in Materials and Methods. Each point in B and C represents the mean value of two or five independent experiments, respectively, using 3–7.5-μl equivalents of cytosol per assay. The maximum amounts of 3H incorporated into VSV-G protein were 9,735, 11,682 ± 2,159, and 3,985 ± 980 cpm in for the medial, trans, and TGN assays, respectively.
Figure 9
ARF depletion has no effect on the NEM sensitivity of donor and acceptor membranes. Donor (open symbols) or acceptor (closed symbols) membranes were pretreated separately with NEM, as described in Materials and Methods. The medial (A), trans (B), and TGN (C) assays were analyzed with either unfractionated cytosol containing ARF (circles) or ARF-depleted cytosol (squares) at 2.5-μl equivalents per assay. The control values for assays with unfractionated cytosol were 6,724 ± 1, 11,457 ± 923, and 3,461 ± 123 cpm 3H incorporated into VSV-G protein in the medial, trans, and TGN assays, respectively. For the ARF-depleted cytosol, they were 5,433 ± 298, 7,750 ± 354, and 2,562 ± 37 cpm.
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