Changes in cellular distribution regulate SKD1 ATPase activity in response to a sudden increase in environmental salinity in halophyte ice plant

Abstract

Halophyte Mesembryanthemum crystallinum L. (ice plant) rapidly responds to sudden increases in salinity in its environment by activating specific salt-tolerant mechanisms. One major strategy is to regulate a series of ion transporters and proton pumps to maintain cellular Na⁺/K⁺ homeostasis. Plant SKD1 (suppressor of K⁺ transport growth defect 1) proteins accumulate in cells actively engaged in the secretory processes, and play a critical role in intracellular protein trafficking. Ice plant SKD1 redistributes from the cytosol to the plasma membrane hours after salt stressed. In combination with present knowledge of this protein, we suggest that stress facilitates SKD1 movement to the plasma membrane where ADP/ATP exchange occurs, and functions in the regulation of membrane components such as ion transporters to avoid ion toxicity.

SKD1 is an AAA-type ATPase that catalyzes the last step of multivescular bodies (MVBs) sorting. MVBs are a versatile organelle, where exocytic and endocytic pathways meet.¹ The sorting complex on the MVB membrane consists of four ESCRT (endosomal sorting complex required for transport) complexes: ESCRT-0, I, II, and III.² In higher plants, most of the ESCRT components and their interacting partners have been identified.³ Potential ESCRT cargo proteins include plasma membrane-bound auxin carriers PINFORMED1 (PIN1), PIN2, and AUXIN-RESISTANT1 (AUX1).⁴ In mammalian cells, the ESCRT machinery is involved in the downregulation of several activated growth factor receptors.⁵ For example, epidermal growth factor (EGF), which stimulates cell proliferation, binds to an EGF receptor (EGFR) on the plasma membrane and activates a downstream signal transduction cascade. The activated EGF-EGFR complexes are then internalized, sorted in MVBs by ESCRT machinery, and delivered to lysosomes for degradation.⁶ In yeast, P-type sodium pump ENA1 is also a potential target for ESCRT machinery.⁷

McSKD1 is a salt-induced gene identified from halophyte Mesembryanthemum crystallinum L.;⁸ reduced SKD1 expression renders Arabidopsis more salt sensitive.⁹ McSKD1 is enriched in microsomal fractions and distributes partially in the plasma membrane and partially in the punctate structure of the cytosol.¹⁰ The distribution of SKD1 rapidly changes such that about 80% of SKD1 appears around the cell surface after 6 h of salt stress.¹¹ We collected cells treated with 200 mM NaCl for various lengths of time and used an AP-conjugated secondary antibody to stain cells. After examining a large number of cells at different time points, we found the punctate structure began to aggregate into small vesicles 30 min after salt stress and redistributed to the cell surface within two hours. To confirm the localization of SKD1, we used anti-HKT2 (high affinity K⁺ transporter 2),¹² a plasma membrane marker, together with anti-SKD1 to dual-label ice plant root cells (Fig. 1). Double-labeling immunofluorescence showed SKD1 protein scattered throughout the cytoplasm (green fluorescence), while HKT2 protein localized primarily in the plasma membrane (red fluorescence). Merging the images showed the plasma membrane as the site of SKD1 and HKT2 colocalization (an estimated 20%). Upon addition of 200 mM NaCl for 6 h, 55% colocalization of SKD1 with the plasma membrane marker was observed (Fig. 1). The results clearly indicate that increased salinity induced the redistribution of SKD1 from the cytoplasm to the plasma membrane.

Figure 1. Immunofluorescence detection of cellular localization of SKD1 with the plasma membrane marker HKT2. Root cells isolated from 3-d-old ice plant seedlings (control) or treated with 200 mM NaCl for 6 h (salt) were dual labeled by anti-SKD1 and anti-HKT2. Confocal images represent single images of Cy3/Alexa 488 dual labeling of a representative cell. Green fluorescence indicates SKD1 image while HKT2 is shown by red fluorescence. The presence of yellow in the merged image indicates the colocalization area of the two proteins. Numbers on the right indicate the percentage of overlapping area (top: yellow/red; bottom: yellow/green). At least 10 cells were calculated in each treatment. All Bars = 5 μm

AAA-type ATPases are molecular motors that drive energy from ATP hydrolysis to disassemble complexes, and unfold or unwind macromolecules, similar to the function of G proteins.¹³ The crystal structure of human SKD1 showed they oligomerize into a double-layer, hexameric ring structure upon ATP binding.¹⁴ In the absence of ATP, SKD1 appears primarily in the form of dimer. Their results confirmed a functional model of SKD1 proposed by Babst and colleagues,¹⁵ where SKD1 proteins cycle dynamically between cytosolic ADP-bound dimers and membrane-associated ATP-charged oilgomers. We found ADP to be a potent inhibitor of ATPase activity of SKD1, whereby the addition of an equal molarity of ADP in the assay decreased the SKD1 activity by 50%, while addition of unlabeled ATP only decreased its activity by 15%.¹⁰ Tight binding by ADP leading to the inhibition of ATPase activity was confirmed by a nucleotide-binding assay.¹⁰ Change in nucleotide-binding state from an ADP- to an ATP-bound state plays an essential role in oligomerization and membrane localization of SKD1.¹⁶ Although a nucleotide exchange step has been proposed in the SKD1 cycling model,¹⁵ the detail mechanism of ADP/ATP exchange remains unknown.

SKD1 interacts with ESCRT-III components VPS2, VPS20, and VPS24 through the N-terminal MIT domain.^17-19 SKD1 interacts with Vta1/LIP5 through the C-terminal β domain to reinforce a hexameric ring structure and activate ATPase activity.²⁰ In addition to ESCRT-related proteins, SKD1 also interacts with enzymes catalyzing post-translational modifications.¹¹ A ser/thr protein kinase SnRK1 (sucrose non-fermenting 1-related kinase 1) interacts with the N- and C-terminus of SKD1, and catalyzes phosphorylation of SKD1. A plasma membrane-bound RING-type E3 ligase CPN1 (copine 1) also interacts with the C-terminus of SKD1, and catalyzes ubiquitination of SKD1. Ice plant SKD1 and SnRK1 interact with each other in the cytosol under normal growth conditions. Upon salt stress, the SKD1-SnRK1 complex redistributes toward the cell surface, where CPN1 resides.¹¹ Figure 1 illustrates that an increased amount of SKD1 is associated with the plasma membrane at early stages of salt stress. We propose that post-translational modifications of SKD1 facilitate its flux toward the plasma membrane, potentially increasing the rate of ADP/ATP exchange, and subsequently increasing the assembly and catalytic activity of SKD1 (Fig. 2). The ATP-charged SKD1 oligomer provides energy to ESCRT-dependent and ESCRT-independent machineries that are involved in the response to salt stress in this halophyte.

Figure 2. Model for the ADP/ATP exchange of SKD1 on the plasma membrane. Under normal growth condition, cycling between ADP-bound (ellipse) and ATP-bound (rectangle) forms of SKD1 is balanced dynamically. Under high salinity condition, cells invest lots of ATP to maintain Na⁺/K⁺ homeostasis and activate stress responses. The rate of ADP/ATP exchange on the plasma membrane may be limited by the supply of ATP at early stages of salt stress. MVB, multivescular bodies; ESCRT-III, complex III of endosomal sorting complex required for transport

Glossary

Abbreviations:

HKT2

high affinity K⁺ transporter 2

MVB

multivescular bodies

SKD1

suppressor of K⁺ transport growth defect 1

ESCRT

endosomal sorting complex required for transport