Dietmar Gerald Mehlhorn

Biological processes in eukaryotes depend on the spatio-temporal compartmentalization of their cells. Integrity and positioning of organelles on the other hand rely on the organization of the actin cytoskeleton. Previously, it has been shown that changes of the plants largest organelle, the vacuole, depend on a functional actin organization. The connection between actin filaments and the vacuole is established by the family of Networked (NET) 4 proteins and, consequently, altering NET4 abundance impacts vacuolar morphology. However, the precise regulatory mechanism is unknown and gene deletions of NET4 did not result in a global growth phenotype. Here, we show that NET4 functions redundantly with NET3, interacting with RABG3-GTPases at the vacuole to allow for homotypic fusion or, alternatively, the generation of endoplasmic reticulum (ER) - vacuole contact sites. We found that ER-resident NET3 is able to interact with RABG3 residing at the tonoplast and that NET4 interacts with the contact site protein VAP27-1 at the ER. Generation of net3 net4 triple mutants by CRSIPR-guided mutagenesis helped us to overcome functional redundancy, resulting in impaired plant growth and development. Our results demonstrate how diversification of NET genes led to functional redundancy between different family members to create cellular plasticity of vascular plants. We hypothesize that establishment of a direct ER-vacuole connection enables direct lipid and protein transfer which is especially important in young and fast-growing cells. Availability of lipids would facilitate rapidly expanding vacuoles, which are the basis for high cell elongation rates and eventually fast plant growth.

SUMMARY: The final step in secretion is membrane fusion facilitated by SNARE proteins that reside in opposite membranes. The formation of a trans‐SNARE complex between one R and three Q coiled‐coiled SNARE domains drives the final approach of the membranes providing the mechanical energy for fusion. Biological control of this mechanism is exerted by additional domains within some SNAREs. For example, the N‐terminal Longin domain (LD) of R‐SNAREs (also called Vesicle‐associated membrane proteins, VAMPs) can fold back onto the SNARE domain blocking interaction with other cognate SNAREs. The LD may also determine the subcellular localization via interaction with other trafficking‐related proteins. Here, we provide cell‐biological and genetic evidence that phosphorylation of the Tyrosine57 residue regulates the functionality of VAMP721. We found that an aspartate mutation mimics phosphorylation, leading to protein instability and subsequent degradation in lytic vacuoles. The mutant SNARE also fails to rescue the defects of vamp721vamp722 loss‐of‐function lines in spite of its wildtype‐like localization within the secretory pathway and the ability to interact with cognate SNARE partners. Most importantly, it imposes a dominant negative phenotype interfering with root growth, normal secretion and cytokinesis in wildtype plants generating large aggregates that mainly contain secretory vesicles. Non‐phosphorylatable VAMP721^Y57F needs higher gene dosage to rescue double mutants in comparison to native VAMP721 underpinning that phosphorylation modulates SNARE function. We propose a model where short‐lived phosphorylation of Y57 serves as a regulatory step to control VAMP721 activity, favoring its open state and interaction with cognate partners to ultimately drive membrane fusion.

Insertion of membrane proteins into the lipid bilayer is a crucial step during their biosynthesis. Eukaryotic cells face many challenges in directing these proteins to their predestined target membrane. The hydrophobic signal peptide or transmembrane domain (TMD) of the nascent protein must be shielded from the aqueous cytosol and its target membrane identified followed by transport and insertion. Components that evolved to deal with each of these challenging steps range from chaperones to receptors, insertases, and sophisticated translocation complexes. One prominent translocation pathway for most proteins is the signal recognition particle (SRP)-dependent pathway which mediates co-translational translocation of proteins across or into the endoplasmic reticulum (ER) membrane. This textbook example of protein insertion is stretched to its limits when faced with secretory or membrane proteins that lack an amino-terminal signal sequence or TMD. Particularly, a large group of so-called tail-anchored (TA) proteins that harbor a single carboxy-terminal TMD require an alternative, post-translational insertion route into the ER membrane. In this review, we summarize the current research in TA protein insertion with a special focus on plants, address challenges, and highlight future research avenues.

Significance: The GET pathway is required for the insertion of tail-anchored (TA) membrane proteins in the endoplasmic reticulum (ER) of yeast and mammals. Some orthologous genes had also been identified in higher plants with the exception of one of the two ER membrane receptors required for membrane insertion. Get2/CAML is required for the pathway’s cytosolic chaperone to dock and release its TA protein cargo. Here we report the identification of the elusive plant GET pathway receptor through an interaction screen inArabidopsis. The candidate allows detection of further Get2/CAML orthologs in higher plants, revealing conservation and function of structural features across kingdoms. Additionally, our results demonstrate that these features, rather than sequence conservation, determine functionality of the candidate within the pathway.

Tail-anchored (TA) proteins are anchored to their corresponding membrane via a single transmembrane segment (TMS) at their C-terminus. In yeast, the targeting of TA proteins to the endoplasmic reticulum (ER) can be mediated by the guided entry of TA proteins (GET) pathway, whereas it is not yet clear how mitochondrial TA proteins are targeted to their destination. It has been widely observed that some mitochondrial outer membrane (MOM) proteins are mistargeted to the ER when overexpressed or when their targeting signal is masked. However, the mechanism of this erroneous sorting is currently unknown. In this study, we demonstrate the involvement of the GET machinery in the mistargeting of suboptimal MOM proteins to the ER. These findings suggest that the GET machinery can, in principle, recognize and guide mitochondrial and non-canonical TA proteins. Hence, under normal conditions, an active mitochondrial targeting pathway must exist that dominates the kinetic competition against other pathways.

Protein–protein interactions (PPIs) play vital roles in all subcellular processes and a number of tools have been developed for their detection and analysis. Each method has its unique set of benefits and drawbacks that need to be considered prior to their application. In fact, researchers are spoilt for choice when it comes to deciding which method to use for the initial detection of a PPI, and which to corroborate the findings. With constant improvements in microscope development, the possibilities of techniques to study PPIs in vivo, and in real time, are continuously enhanced, and expanded. Here, we describe three common approaches, their recent improvements incorporating a 2in1-cloning approach, and their application in plant cell biology: ratiometric Bimolecular Fluorescence Complementation (rBiFC), FRET Acceptor Photobleaching (FRET-AB), and Fluorescent Lifetime Imaging (FRET-FLIM), using Nicotiana benthamiana leaves and Arabidopsis thaliana cell culture protoplasts as transient expression systems.

Significance: Root hairs are unicellular extensions of the rhizodermis, providing anchorage and an increase in surface area for nutrient and water uptake. Their fast, tip-focused growth showcases root hairs as an excellent genetic model to study physiological and developmental processes on the cellular level. We uncovered a root hair phenotype that is dependent on putative Arabidopsis orthologs of the Guided Entry of Tail-anchored (TA) proteins (GET) pathway, which facilitates membrane insertion of TA proteins in yeast and mammals. We found that plants have evolved multiple paralogs of specific GET pathway components, albeit in a compartment-specific manner. In addition, we show that differential expression of pathway components causes pleiotropic growth defects, suggesting alternative pathways for TA insertion and additional functions of GET in plants.