Adaptor Protein-3-Dependent Vacuolar Trafficking Involves a Subpopulation of COPII and HOPS Tethering Proteins

Abstract

ORCID IDs: 0000-0001-6345-6855 (J.-G.W.); 0000-0002-3501-5857 (Y.Z.).Plant vacuoles are versatile organelles critical for plant growth and responses to environment. Vacuolar proteins are transported from the endoplasmic reticulum via multiple routes in plants. Two classic routes bear great similarity to other phyla with major regulators known, such as COPII and Rab5 GTPases. By contrast, vacuolar trafficking mediated by adaptor protein-3 (AP-3) or that independent of the Golgi has few recognized cargos and none of the regulators. In search of novel regulators for vacuolar trafficking routes and by using a fluorescence-based forward genetic screen, we demonstrated that the multispan transmembrane protein, Arabidopsis (Arabidopsis thaliana) PROTEIN S-ACYL TRANSFERASE10 (PAT10), is an AP-3-mediated vacuolar cargo. We show that the tonoplast targeting of PAT10 is mediated by the AP-3 complex but independent of the Rab5-mediated post-Golgi trafficking route. We also report that AP-3-mediated vacuolar trafficking involves a subpopulation of COPII and requires the vacuolar tethering complex HOPS. In addition, we have identified two novel mutant alleles of AP-3d, whose point mutations interfered with the formation of the AP-3 complex as well as its membrane targeting. The results presented here shed new light on the vacuolar trafficking route mediated by AP-3 in plant cells.

AP3M harbors actin filament binding activity that is crucial for vacuole morphology and stomatal closure in Arabidopsis

“…We complemented the ap-3m-3 mutant expressing 35S:GFP-SUC4 with the wild-type 5.0-kb AP3M genomic fragment (COM AP3M ) or the 5.0-kb AP3M genomic fragment harboring the 3A mutation (COM AP3M-3A ) and observed the localization of GFP-SUC4. Consistent with previous findings (27,28), the tonoplast localization of SUC4 was abolished in the ap-3m-3 mutant ( Fig. 7C and SI Appendix, Fig.…”

“…Studies in mammalian cells have revealed that: 1) The β3 subunit of the AP3 complex interacts with the clathrin heavy chain via its C domain (21); 2) the δ-subunit is important for the recruitment of this complex to the membrane (20), while the δ-subunit interacts with the σ-subunit to recognize dileucine-based sorting signals (DE[XXXL]LI) (22); and 3) the μ-subunit binds to tyrosine-based sorting signals (YXXΦ) (23). In plants, defects in the AP3 complex lead to aberrant vacuole morphology (24)(25)(26) and abnormal protein trafficking, with most protein retained in the Golgi apparatus, such as SUC4 (27) and PAT10 (28), in root epidermal cells and embryo cells. Therefore, the AP3 complex is crucial for plant development and various cellular activities, especially for the proper functioning of the Significance Actin filament dynamics and the proper functioning of adaptor complexes are required for the sorting of cargo proteins from the Golgi to the vacuole, but the relationship between these 2 important processes remains unclear.…”

“…vacuole (24)(25)(26)(27)(28)(29)(30). However, whether AP3 is involved in regulating stomatal movement remains unclear.…”

Arabidopsis Chloroplast protein for Growth and Fertility1 (CGF1) and CGF2 are essential for chloroplast development and female gametogenesis

“…For constructs used in subcellular localization, the coding sequences of CGF1, CGF2, and various mutant forms were amplified with the following primer pairs: ZP4997/ ZP4998 for CGF1, ZP5738/ZP5739 for CGF2, ZP4997/ ZP9786 for CGF1 SP , ZP5738/ZP9784 for CGF2 SP , ZP9787/ZP4998 for CGF1 ΔSP , and ZP9785/ZP5739 for CGF2 ΔSP . The resultant entry vectors were used in LR reactions with the destination vector Pro UBQ10 :GW-GFP [24] or Pro 35S :GW-GFP [25] to generate expression vectors, including Pro UBQ10 :CGF1-GFP, Pro UBQ10 :CGF2-GFP, Pro 35S :CGF1 SP -GFP, Pro 35S :CGF2 SP -GFP, Pro 35S :CGF1 ΔSP -GFP, and Pro 35S :CGF2 ΔSP -GFP.…”

The Plant Trans-Golgi Network: Not Just a Matter of Distinction

“…More recently, TGN-localized AP1 g-adaptins were found to mediate the targeting of membrane proteins with di-Leu motifs to the tonoplast (Wang et al, 2014). Intriguingly, Arabidopsis AP3 has been implicated in a TGN-bypass pathway that transfers cargo directly from Golgi cisternae to the vacuole and is facilitated by the HOPS-tethering complex (Feraru et al, 2010;Zwiewka et al, 2011;Feng et al, 2017). Perhaps with the exception of AP2, which operates in clathrin-mediated endocytosis at the plasma membrane (Krauss et al, 2006;Di Rubbo et al, 2013), a function in vacuolar transport appears to be the norm among Arabidopsis AP complexes.…”