Abstract
Plant cells perceive and adapt to an ever-changing environment by modifying their plasma membrane (PM) proteome. Whereas secretion deposits new integral membrane proteins, internalization by endocytosis removes membrane proteins and associated ligands, largely with the aid of adaptor protein (AP) complexes and the scaffolding molecule clathrin. Two AP complexes function in clathrin-mediated endocytosis at the PM in plant cells, the heterotetrameric AP-2 complex and the hetero-octameric TPLATE complex (TPC). Whereas single subunit mutants in AP-2 develop into viable plants, genetic mutation of a single TPC subunit causes fully penetrant male sterility and silencing single subunits leads to seedling lethality. To address TPC function in somatic root cells, while minimizing indirect effects on plant growth, we employed nanobody-dependent delocalization of a functional, GFP-tagged TPC subunit, TML, in its respective homozygous genetic mutant background. In order to decrease the amount of functional TPC at the PM, we targeted our nanobody construct to the mitochondria and fused it to TagBFP2 to visualize it independently of its bait. We furthermore limited the effect of our delocalization to those tissues that are easily accessible for live-cell imaging by expressing it from the PIN2 promoter, which is active in root epidermal and cortex cells. With this approach, we successfully delocalized TML from the PM. Moreover, we also show co-recruitment of TML-GFP and AP2A1-TagRFP to the mitochondria, suggesting that our approach delocalized complexes, rather than individual adaptor complex subunits. In line with the specific expression domain, we only observed minor effects on root growth, yet realized a clear reduction of endocytic flux in epidermal root cells. Nanobody-dependent delocalization in plants, here exemplified using a TPC subunit, has the potential to be widely applicable to achieve specific loss-of-function analysis of otherwise lethal mutants.
Highlights
Cells are delineated by their plasma membrane (PM)
To verify that the GFP nanobody did not interfere with the mitochondrial targeting, we expressed it under the control of the 35Sprom together with the red mitochondrial mt-rk marker (Nelson et al, 2007) in N. benthamiana abaxial leaf epidermal cells and visualized both fusion proteins using spinning disk confocal microscopy
Analyzing how impaired TPLATE complex (TPC) function directly affects endocytosis is hampered by the male sterility and/or seedling lethal mutant phenotypes following genetic interference of individual subunits (Gadeyne et al, 2014)
Summary
The PM houses a plethora of proteins ranging from receptors and ion channels to structural membrane proteins. Many of these PM proteins, commonly termed cargo, are responsible for cellular communication with the outside world. Endocytosis is the cellular process where cargoes, associated ligands as well as lipids are internalized from the PM. Endocytosis thereby provides a way to regulate the content and modulate protein activity at the PM. A predominant and well-studied form of endocytosis is clathrin-mediated endocytosis (CME; Bitsikas et al, 2014). CME refers to the dependency of the scaffolding protein clathrin, which coats the developing and mature vesicles (Robinson, 2015).
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