ESCRT components ISTL1 andLIP5 are required for tapetal function and pollen viability.

Pollen wall assembly is crucial for pollen development and plant fertility. The durable biopolymer sporopollenin and the constituents of the tryphine coat are delivered to developing pollen grains by the highly coordinated secretory activity of the surrounding tapetal cells. The role of membrane trafficking in this process, however, is largely unknown. In this study, we used Arabidopsis thaliana to characterize the role of two late-acting endosomal sorting complex required for transport (ESCRT) components, ISTL1 and LIP5, in tapetal function. Plants lacking ISTL1 and LIP5 form pollen with aberrant exine patterns, leading to partial pollen lethality. We found that ISTL1 and LIP5 are required for exocytosis of plasma membrane and secreted proteins in the tapetal cells at the free microspore stage, contributing to pollen wall development and tryphine deposition. Whereas the ESCRT machinery is well known for its role in endosomal trafficking, the function of ISTL1 and LIP5 in exocytosis is not a typical ESCRT function. The istl1 lip5 double mutants also show reduced intralumenal vesicle concatenation in multivesicular endosomes in both tapetal cells and developing pollen grains as well as morphological defects in early endosomes/trans-Golgi networks, suggesting that late ESCRT components function in the early endosomal pathway and exocytosis.

Nonselective Chemical Inhibition of Sec7 Domain-Containing ARF GTPase Exchange Factors.

Small GTP-binding proteins from the ADP-ribosylation factor (ARF) family are important regulators of vesicle formation and cellular trafficking in all eukaryotes. ARF activation is accomplished by a protein family of guanine nucleotide exchange factors (GEFs) that contain a conserved catalytic Sec7 domain. Here, we identified and characterized Secdin, a small-molecule inhibitor of Arabidopsis thaliana ARF-GEFs. Secdin application caused aberrant retention of plasma membrane (PM) proteins in late endosomal compartments, enhanced vacuolar degradation, impaired protein recycling, and delayed secretion and endocytosis. Combined treatments with Secdin and the known ARF-GEF inhibitor Brefeldin A (BFA) prevented the BFA-induced PM stabilization of the ARF-GEF GNOM, impaired its translocation from the Golgi to the trans-Golgi network/early endosomes, and led to the formation of hybrid endomembrane compartments reminiscent of those in ARF-GEF-deficient mutants. Drug affinity-responsive target stability assays revealed that Secdin, unlike BFA, targeted all examined Arabidopsis ARF-GEFs, but that the interaction was probably not mediated by the Sec7 domain because Secdin did not interfere with the Sec7 domain-mediated ARF activation. These results show that Secdin and BFA affect their protein targets through distinct mechanisms, in turn showing the usefulness of Secdin in studies in which ARF-GEF-dependent endomembrane transport cannot be manipulated with BFA.

Arabidopsis ALIX is required for the endosomal localization of the deubiquitinating enzyme AMSH3.

Ubiquitination is a signal for various cellular processes, including for endocytic degradation of plasma membrane cargos. Ubiquitinating as well as deubiquitinating enzymes (DUBs) can regulate these processes by modifying the ubiquitination status of target protein. Although accumulating evidence points to the important regulatory role of DUBs, the molecular basis of their regulation is still not well understood. Associated molecule with the SH3 domain of signal transduction adaptor molecule (STAM) (AMSH) is a conserved metalloprotease DUB in eukaryotes. AMSH proteins interact with components of the endosomal sorting complex required for transport (ESCRT) and are implicated in intracellular trafficking. To investigate how the function of AMSH is regulated at the cellular level, we carried out an interaction screen for the Arabidopsis AMSH proteins and identified the Arabidopsis homolog of apoptosis-linked gene-2 interacting protein X (ALIX) as a protein interacting with AMSH3 in vitro and in vivo. Analysis of alix knockout mutants in Arabidopsis showed that ALIX is essential for plant growth and development and that ALIX is important for the biogenesis of the vacuole and multivesicular bodies (MVBs). Cell biological analysis revealed that ALIX and AMSH3 colocalize on late endosomes. Although ALIX did not stimulate AMSH3 activity in vitro, in the absence of ALIX, AMSH3 localization on endosomes was abolished. Taken together, our data indicate that ALIX could function as an important regulator for AMSH3 function at the late endosomes.

Role of SKD1 Regulators LIP5 and IST1-LIKE1 in Endosomal Sorting and Plant Development.

SKD1 is a core component of the mechanism that degrades plasma membrane proteins via the Endosomal Sorting Complex Required for Transport (ESCRT) pathway. Its ATPase activity and endosomal recruitment are regulated by the ESCRT components LIP5 and IST1. How LIP5 and IST1 affect ESCRT-mediated endosomal trafficking and development in plants is not known. Here we use Arabidopsis mutants to demonstrate that LIP5 controls the constitutive degradation of plasma membrane proteins and the formation of endosomal intraluminal vesicles. Although lip5 mutants were able to polarize the auxin efflux facilitators PIN2 and PIN3, both proteins were mis-sorted to the tonoplast in lip5 root cells. In addition, lip5 root cells over-accumulated PIN2 at the plasma membrane. Consistently with the trafficking defects of PIN proteins, the lip5 roots showed abnormal gravitropism with an enhanced response within the first 4 h after gravistimulation. LIP5 physically interacts with IST1-LIKE1 (ISTL1), a protein predicted to be the Arabidopsis homolog of yeast IST1. However, we found that Arabidopsis contains 12 genes coding for predicted IST1-domain containing proteins (ISTL1-12). Within the ISTL1-6 group, ISTL1 showed the strongest interaction with LIP5, SKD1, and the ESCRT-III-related proteins CHMP1A in yeast two hybrid assays. Through the analysis of single and double mutants, we found that the synthetic interaction of LIP5 with ISTL1, but not with ISTL2, 3, or 6, is essential for normal plant growth, repression of spontaneous cell death, and post-embryonic lethality.

Imaging Plant Cells by High-Pressure Freezing and Serial block-face scanning electron microscopy.

This chapter describes methods to enhanced contrast of plant material processed by high-pressure freezing and freeze substitution for improved visualization by serial block-face scanning electron microscopy (SBEM). The contrast enhancing steps are based on a protocol involving the sequential incubation of samples in heavy metals and sodium thiocarbohydrazide (OTO staining). We also describe the pipeline for imaging plant tissues in a commercial SBEM system (Gatan 3View®) and routines for the image analysis and three-dimensional reconstructions using open-source and commercial software packages.

ESCRT-mediated vesicle concatenation in plant endosomes.

Ubiquitinated plasma membrane proteins (cargo) are delivered to endosomes and sorted by endosomal sorting complex required for transport (ESCRT) machinery into endosome intralumenal vesicles (ILVs) for degradation. In contrast to the current model that postulates that ILVs form individually from inward budding of the endosomal limiting membrane, plant ILVs form as networks of concatenated vesicle buds by a novel vesiculation mechanism. We ran computational simulations based on experimentally derived diffusion coefficients of an ESCRT cargo protein and electron tomograms of Arabidopsis thaliana endosomes to measure cargo escape from budding ILVs. We found that 50% of the ESCRT cargo would escape from a single budding profile in 5-20 ms and from three concatenated ILVs in 80-200 ms. These short cargo escape times predict the need for strong diffusion barriers in ILVs. Consistent with a potential role as a diffusion barrier, we find that the ESCRT-III protein SNF7 remains associated with ILVs and is delivered to the vacuole for degradation.

Endocytosis and Endosomal Trafficking in Plants.

Endocytosis and endosomal trafficking are essential processes in cells that control the dynamics and turnover of plasma membrane proteins, such as receptors, transporters, and cell wall biosynthetic enzymes. Plasma membrane proteins (cargo) are internalized by endocytosis through clathrin-dependent or clathrin-independent mechanism and delivered to early endosomes. From the endosomes, cargo proteins are recycled back to the plasma membrane via different pathways, which rely on small GTPases and the retromer complex. Proteins that are targeted for degradation through ubiquitination are sorted into endosomal vesicles by the ESCRT (endosomal sorting complex required for transport) machinery for degradation in the vacuole. Endocytic and endosomal trafficking regulates many cellular, developmental, and physiological processes, including cellular polarization, hormone transport, metal ion homeostasis, cytokinesis, pathogen responses, and development. In this review, we discuss the mechanisms that mediate the recognition and sorting of endocytic and endosomal cargos, the vesiculation processes that mediate their trafficking, and their connection to cellular and physiological responses in plants.