PtK1 Cells Contain a Nonditfusible, Dominant Factor That Makes the Golgi Apparatus Resistant to Brefeldin A

Brefeldin A (BFA) was shown in earlier studies of numerous cell types to inhibit secretion, induce enzymes of the Golgi stacks to redistribute into the ER, and to cause the Golgi cisternae to disappear. Here, we demonstrate that the PtKt line of rat kangaroo kidney cells is resistant to BFA. The drug did not disrupt the morphology of the Golgi complex in PtKt cells, as judged by immunofluorescence using antibodies to 58(58K) and l l0-kD (B-COP) Golgi proteins, and by fluorescence microscopy of live cells labeled with C6-NBD-ceramide. In addition, BFA did not inhibit protein secretion, not alter the kinetics or extent of glycosylation of the vesicular stomatitis virus (VSV) glycoprotein (G-protein) in VSV-infected PtKt cells. To explore the mechanism of resistance to BFA, PtKt cells were fused with BFA-sensitive CV-1 cells that had been infected with a recombinant SV-40 strain containing the gene for VSV G-protein and, at various times following fusion, the cultures were exposed to BFA. Shortly after cell fusion, heterokaryons contained one Golgi complex associated with each nucleus. Golgi membranes derived from CV-1 cells were sensitive to BFA, whereas those of PtKt origin were BFA resistant. A few hours after fusion, most heterokaryons contained a single, large Golgi apparatus that was resistant to BFA and contained CV-1 galactosyltransferase. In unfused cells that had been perforated using nitrocellulose filters, retention of/3-COP on the Golgi was optimal in the presence of cytosol, ATP, and GTE In perforated cell models of the BFAsensitive MA104 line, BFA caused 3-COP to be released from the Golgi complex in the presence of nucleotides, and either MA104 or PtK~ cytosol. In contrast, when perforated PtKl cells were incubated with BFA, nucleotides, and cytosol from either cell type, ~-COP remained bound to the Golgi complex. We conclude that PtKl cells contain a nondiffusible factor, which is located on or very close to the Golgi complex, and confers a dominant resistance to BFA. It is possible that this factor is homologous to the target of BFA in cells that are sensitive to the drug. T rIE Golgi apparatus comprises a series of membranous compartments in which proteins are modified posttranslationally and sorted so that each is targeted to an appropriate cellular membrane. To fulfill these tasks, the Golgi complex is divided into several structurally and functionally distinct regions through which proteins successively pass: the cis-, medial-, and trans-Golgi cisternae, and the trans-Golgi network (Farquhar, 1985; Griffiths and Simons, 1986). Maintaining the structural integrity of the Golgi complex and its various compartments represents a significant challenge for the cell because large quantities of protein and lipid regularly pass between the Golgi complex and other membranous organelles. For example, proteins destined for processing in the Golgi network are exported from the ER along with more numerous resident ER proteins. This process occurs by a mechanism that concentrates the former proteins 5-10-fold in the Golgi complex, while efficiently returning the ER proteins to their normal place of residence (Green et al., 1981; Griffiths et al., 1984; Warren 1987; Pelham, 1989). An equally important feature of this process is that proteins which perform their functions in the Golgi complex are selectively retained within the Golgi complex. Thus, the exchange of membrane components between the ER and the Golgi membrane is opposed by at least two concentration gradients, one favoring escape of abundant ER proteins into Golgi membranes that lack them, and another favoring absorption of Golgi membrane constituents into the ER. Evidently, the cell uses multiple sorting steps to prevent the net transfer of ER proteins to the Golgi membrane and of Golgi proteins to the ER. One of these sorting steps is accomplished during the formation of transport vesicles that emanate from the ER (Palade, 1975; Bergmann and Singer, 1983), while another occurs in an immunologically distinct compartment juxtaposed to the Golgi complex (Seraste and Kuismanen, 1984; Tooze et al., 1984; Warren, 1987; Schweizer et al., 1988; Pelham, 1989). This intermediate compartment has been proposed to segregate "escaped" ER proteins from proteins which proceed through the secretory pathway. As a result, component proteins of the ER can be recycled © The Rockefeller University Press, 0021-9525/91/06/1009/15 $2.00 The Journal of Cell Biology, Volume 113, Number 5, June 1991 1009-1023 1009 on July 9, 2017 jcb.rress.org D ow nladed fom from the intermediate compartment back to the ER, while proteins that require further posttranslational modifications can be transported to cis-Golgi cisternae. It seems likely that these protein sorting events are coupled to the formation of vesicles derived from the intermediate compartment, and that some of the vesicles fuse selectively with the ER, while others merge specifically with the cis-Golgi membrane (reviewed in Pelham, 1989). In principle, such a mechanism could prevent the flow of Golgi membranes into the intermediate compartment, and by extension, into the ER. Further insight into the mechanism for preventing Golgi proteins from escaping into the ER has been gained recently through studies of the fungal metabolite, brefeldin A (BFA) t. Treatment of cultured cells with BFA was found to cause resident Golgi proteins to redistribute into the ER by a mechanism that requires energy and intact microtubules (Lippincott-Schwartz et al., 1990). Moreover, the application of BFA to cultured cells has provided evidence that the pathway for membrane flow between the ER and Golgi membrane contains the aforementioned ER recycling arm, which is otherwise difficult to detect (Lippincott-Schwartz et al., 1990). Based on these and related findings (Fujiwara et al., 1988; Doms et al., 1989; Ulmer and Palade, 1989; Lippincott-Schwartz et al., 1990), it has been proposed that BFA acts through a specific molecular target and directly inhibits only one aspect of membrane traffic (Lippincott-Schwartz et al., 1990). If BFA does, indeed, inactivate a specific Golgi target that plays a critical role in maintaining the separate identity of Golgi membranes, then BFA-treated cells may be as useful for understanding the control of intracellular protein traffic in animal cells as mutant strains have been for unravelling the secretory pathway in yeast. As of now, however, the hypothesis that BFA acts directly on a single intracellular target and exerts a limited number of specific effects has been supported mainly by the observations that BFA does not alter cellular ATP levels, protein synthesis, or endocytosis, and affects cells at submicromolar concentrations (Misumi et al., 1986; Lippincott-Schwartz et al., 1990). The identification of "mutant" cells containing a Golgi complex resistant to BFA could provide a means for identifying the hypothetical BFA target, and for determining whether the membrane redistribution observed in cells treated with the drug depends upon the imperfect operation of pathways normally active in the cell or upon novel pathways that are induced by BFA. In this report, we present evidence that the Golgi apparatus in the rat kangaroo kidney cell line, PtKI, is unaffected structurally or functionally by BFA. The resistance of PtK~ cells to BFA cannot be explained by an inability of the drug to enter the ceils, or by the sequestration or inactivation of BFA once it gains access to the cytoplasm. Instead, in PtKt cells the Golgi complex itself, or a compartment directly adjacent to the Golgi apparatus appears to contain a dominant, nondiffusible factor that confers resistance to BFA. This factor may represent a BFA-insensitive version of the molecule which serves as the target for BFA in other cell types. 1. Abbreviations used in this paper: BFA, brefeldin A; endo H, endoglycosidase H; G Protein, glycoprotein; Gai T, galactosyltransferase; VSV, vesicular stomatitis virus. Materials and Methods Biochemical Supplies and Viral Reagents BFA was purchased from Epicentre Technologies (Madison, Wl) and was dissolved at 10 mg/ml (36 raM) in DMSO or ethanol. C6-NBD-ceramide was purchased from Molecular Probes (Eugene, OR) and was dissolved at 5 mM in ethanol. Endoglycosidase H (endo H), ATE GTP, creatine phosphate, and rabbit creatine phosphokinase were purchased from Boehringer Mannheim Biochemicais (Indianapolis, IN). All other chemicals and biochemicals were from Sigma Chemical Co. (St. Louis, MO). Vesicular stomatifis virus (VSV) was propagated in MDCK cells (Roth and Compans, 1981). Recombinant SV-40 virus carrying the gene for VSV G protein was prepared as described previously (Doyle et ai., 1985).