ß-COP mutants show specific high sensitivity to chloride ions

ABSTRACT

Coat Protein I (COPI) consists of a complex (coatomer) formed by seven subunits (α-, β-, β’-, γ-, δ-, ε-, and ζ-COP) that is recruited to Golgi membranes to form vesicles that shuttle from the Golgi apparatus to the ER and between Golgi stacks. Recently, it has been described that loss of function mutants of the two Arabidopsis β-COP genes, β1-COP and β2-COP, showed increased sensitivity to salt stress (NaCl). Using a mixture of either Na⁺ or Cl⁻ salts, we have now found that β-COP mutants are specifically and highly sensitive to chloride ions.

Proteins and lipids are transported between organelles in membrane-bound vesicles in eukaryotic cells. These vesicles are generated by the assembly of protein coats and they are captured at their target organelle by tethering factors. Coat proteins are recruited from the cytosol onto specific organelle membranes and they are important for cargo selection and vesicle formation. Coat protein complex I (COPI) vesicles shuttle from the Golgi apparatus to the endoplasmic reticulum and between Golgi stacks, although the directionality of intra-Golgi COPI vesicles is still controversial. The machinery involved in the formation of COPI vesicles has been characterized and comprises the small G protein ARF1, proteins from the p24 family and the COPI coatomer complex, formed by seven subunits (α-, β-, β’-, γ-, δ-, ε-, and ζ-COP).^1–3 The biogenesis of COPI vesicles starts with the activation of ARF1 at the Golgi apparatus, by GDP/GTP exchange leading to membrane insertion. Membrane-anchored ARF1 then recruits en bloc the coatomer complex, which also interacts with p24 proteins and cargo proteins.⁴ From structural studies, two coatomer subcomplexes can be distinguished, the α-/β’-/ε-COP subcomplex and the β-/γ-/δ-/ζ-COP subcomplex. Nevertheless, coatomer exists as a single complex in the cytosol and is recruited to the Golgi membranes in one single step.^1–3 This contrasts with the stepwise membrane association of other coats and the utility of such way of recruitment for COPI function is not known. It also remains poorly understood how stoichiometric expression of the different subunits is achieved in the assembly.

The coatomer complex is not only part of the machinery involved in the biogenesis of COPI vesicles but it is also required to select the cargo to be included in the vesicles. Indeed, membrane proteins to be included in COPI vesicles can be recognized directly by the coatomer complex through sorting motifs present in their cytosolic sequence, which are recognized by different coatomer subunits (including α-, β-, β’-, γ-, and δ-COP) (Table 1).^1–3

Table 1.

COPI sorting signals and interacting COPI subunits. These data are from mammalian and yeast cells. Dilysine motifs also seem to bind COPI subunits in plant cells as it is the case of the KxD/E motif, ⁵ but the interacting COPI subunits have not been identified. * Φ/Ψ is an aromatic or bulky hydrophobic residue. ** B is basic amino acid

COPI sorting signals	Interacting COPI subunit	References
KKxx, KxKxx	Alfa, Beta´	6–10
[φ/ψ/R)*RxR	Beta , Delta	11
FFxxBB**[x]n in p24 family	Gamma	12

To explore the function of COP-I coat proteins in plants, several loss of function approaches of COPI subunits have been described.^13–15 Recently, we have reported that depletion of β-COP altered the structure of the Golgi apparatus. In particular, we found an increased length of Golgi stacks, which in some cases may be the consequence of lateral fusion between Golgi stacks.¹⁶ A similar Golgi phenotype was observed upon treatment of BY-2 cells with BFA, which prevents COPI vesicle formation.¹⁷ In addition, depletion of β-COP compromised growth and salt tolerance in Arabidopsis.¹⁶ T-DNA insertion single mutants of the two β-COP genes, β1-COP and β2-COP, were hypersensitive to salt stress (NaCl) but not to mannitol, suggesting that the NaCl sensitivity observed in the mutants was not due to osmotic stress and might be caused by ion (Na⁺ and/or Cl⁻) toxicity.

To determine whether β1-cop and β2-cop mutants were specifically sensitive to Cl⁻, they were treated with KCl. As shown in Figure 1, both mutants were extremely sensitive to KCl, in contrast to wild-type. To further assess the individual effect of Cl⁻ ions, β1-cop and β2-cop mutants were treated with a mixture of Cl⁻ salts (20 mM CaCl₂, 20 mM MgCl₂, and 40 mM KCl), at similar electrical conductivity and pH as 120 mM NaCl.^18,19 The results obtained suggest that both mutants are specifically and highly sensitive to chloride ions (Figure 2). In contrast, when both mutants were treated with a mixture of Na⁺ salts (20 mM Na₂SO₄, 20 mM Na₂HPO₄, and 40 mM NaNO₃) no differences were detected compared to wild-type (Figure 2).

Figure 1.

Phenotypic analysis of β1-cop and β2-cop mutants exposed to KCl. Wild type (Col-0) and β1-cop and β2-cop seeds were sown on 0.5 x MS as a control¹⁶ and 0.5 x MS supplemented with 110 mM or 130 mM KCl. Left panel shows an image of a representative experiment. Right panel shows the percentage of seedlings with green cotyledons calculated after 12 d and are mean ± s.e.m. (n = 100) of four independent experiments. Statistical significance: ***p < .001. Scale bar 1.5 cm

Figure 2.

Phenotypic analysis of β1-cop and β2-cop mutants exposed to Cl⁻ or Na⁺ ions. Wild type (Col-0) and β1-cop and β2-cop seeds were sown on 0.5 x MS as a control¹⁶ and 0.5 x MS supplemented with a mixture of 120 mM Cl⁻ salts or 120 mM Na⁺ salts (see text for composition). Top panel shows an image of a representative experiment. Bottom panel shows the percentage of seedlings with true leaves calculated after 12 d and are mean ± s.e.m. (n = 100) of five independent experiments. Statistical significance: ns, not significant; *p < .05; ***p < .001

The chloride toxicity observed in the β1-cop and β2-cop mutants could be due to mislocalization or reduced activity of Cl⁻ channels/transporters. Interestingly, the β-COP subunit has been involved in the regulation of the surface expression of several ion channels in mammalian cells, including chloride channels.²⁰ Depletion of β-COP subunit has been shown to alter trafficking to the plasma membrane of the chloride channels CFRT and ANO1 and, importantly, to change their channel activity in human cells.^21,22 Many of these ion channels have been shown to interact with COPI subunits.^20–23 In addition, it cannot be discarded that the increased Cl⁻ sensitivity observed in the β-COP mutants could be due to the alteration of the structure of the Golgi apparatus, ¹⁶ as many ion channels/transporters have been described to localize in the Golgi apparatus.^24–28

Funding Statement

Support for this research was provided by the Generalitat Valenciana [GVACOMP2014-202] and Ministerio de Economía, Industria y Competitividad, Gobierno de España [BFU2016-76607P].