Sheung Kwan Lam

Rice SCAMP1 Defines Clathrin-Coated, trans-Golgi–Located Tubular-Vesicular Structures as an Early Endosome in Tobacco BY-2 Cells

We recently identified multivesicular bodies (MVBs) as prevacuolar compartments (PVCs) in the secretory and endocytic pathways to the lytic vacuole in tobacco (Nicotiana tabacum) BY-2 cells. Secretory carrier membrane proteins (SCAMPs) are post-Golgi, integral membrane proteins mediating endocytosis in animal cells. To define the endocytic pathway in plants, we cloned the rice (Oryza sativa) homolog of animal SCAMP1 and generated transgenic tobacco BY-2 cells expressing yellow fluorescent protein (YFP)–SCAMP1 or SCAMP1-YFP fusions. Confocal immunofluorescence and immunogold electron microscopy studies demonstrated that YFP-SCAMP1 fusions and native SCAMP1 localize to the plasma membrane and mobile structures in the cytoplasm of transgenic BY-2 cells. Drug treatments and confocal immunofluorescence studies demonstrated that the punctate cytosolic organelles labeled by YFP-SCAMP1 or SCAMP1 were distinct from the Golgi apparatus and PVCs. SCAMP1-labeled organelles may represent an early endosome because the internalized endocytic markers FM4-64 and AM4-64 reached these organelles before PVCs. In addition, wortmannin caused the redistribution of SCAMP1 from the early endosomes to PVCs, probably as a result of fusions between the two compartments. Immunogold electron microscopy with high-pressure frozen/freeze-substituted samples identified the SCAMP1-positive organelles as tubular-vesicular structures at the trans-Golgi with clathrin coats. These early endosomal compartments resemble the previously described partially coated reticulum and trans-Golgi network in plant cells.

A vesicle carrier that mediates peroxisome protein traffic from the endoplasmic reticulum

Pex19p, a soluble cytoplasmic transport protein, is required for the traffic of the peroxisomal membrane proteins Pex3p and Pex15p from the endoplasmic reticulum (ER) to the peroxisome. We documented Pex15p traffic from the ER using a chimeric protein containing a C-terminal glycosylation acceptor peptide. Pex15Gp expressed in wild-type yeast cells is N-glycosylated and functions properly in the peroxisome. In contrast, pex19Δ-mutant cells accumulate the glycoprotein Pex15Gp in the ER. We developed a cell-free preperoxisomal vesicle-budding reaction in which Pex15Gp and Pex3p are packaged into small vesicles in the presence of cytosol, Pex19p, and ATP. Secretory vesicle budding (COPII) detected by the packaging of a SNARE protein (soluble N-ethylmaleimide-sensitive attachment protein receptor) occurs in the same incubation but does not depend on Pex19p. Conversely a dominant GTPase mutant Sar1p which inhibits COPII has no effect on Pex3p packaging. Pex15Gp and Pex3p budded vesicles sediment as low-buoyant-density membranes on a Nycodenz gradient and copurify by affinity isolation using native but not Triton X-100–treated budded vesicles. ER–peroxisome transport vesicles appear to rely on a novel budding mechanism requiring Pex19p and additional unknown factors.

Wortmannin induces homotypic fusion of plant prevacuolar compartments

Wortmannin, a specific inhibitor of phosphatidyl-inositol 3-kinase, is a useful tool for studying protein trafficking and identifying organelles in the plant secretory and endocytic pathways. It has recently been demonstrated that wortmannin at 16.5 μM or 33 μM caused the prevacuolar compartments (PVCs), identified as multivesicular bodies (MVBs) by their enrichment in vacuolar sorting receptor (VSRs) proteins and the BP-80 reporter, to form small vacuoles rapidly. However, the source(s) of the membrane needed for the rapid enlargement of PVCs/MVBs has been unclear. Using both confocal immunofluorescence and immunogold EM with high pressure freeze substitution of plant samples, it has been demonstrated here that wortmannin induces homotypic fusions of PVCs/MVBs thus providing an explanation for the demand for extra membrane. In addition, possible wortmannin-induced fusions between the trans-Golgi network (TGN) and PVC, as well as between the small internal vesicles and PVC membrane, were also observed and they may also contribute to the membranes needed for PVC enlargement. In contrast to mammalian cells and yeast, wortmannin-induced fusion of PVCs appears to be unique to plants.

BFA‐induced compartments from the Golgi apparatus and trans‐Golgi network/early endosome are distinct in plant cells

Brefeldin A (BFA) is a useful tool for studying protein trafficking and identifying organelles in the plant secretory and endocytic pathways. At low concentrations (5-10 microg ml(-1)), BFA caused both the Golgi apparatus and trans-Golgi network (TGN), an early endosome (EE) equivalent in plant cells, to form visible aggregates in transgenic tobacco BY-2 cells. Here we show that these BFA-induced aggregates from the Golgi apparatus and TGN are morphologically and functionally distinct in plant cells. Confocal immunofluorescent and immunogold electron microscope (EM) studies demonstrated that BFA-induced Golgi- and TGN-derived aggregates are physically distinct from each other. In addition, the internalized endosomal marker FM4-64 co-localized with the TGN-derived aggregates but not with the Golgi aggregates. In the presence of the endocytosis inhibitor tyrphostin A23, which acts in a dose- and time-dependent manner, SCAMP1 (secretory carrier membrane protein 1) and FM4-64 are mostly excluded from the SYP61-positive BFA-induced TGN aggregates, indicating that homotypic fusion of the TGN rather than de novo endocytic trafficking is important for the formation of TGN/EE-derived BFA-induced aggregates. As the TGN also serves as an EE, continuously receiving materials from the plasma membrane, our data support the notion that the secretory Golgi organelle is distinct from the endocytic TGN/EE in terms of its response to BFA treatment in plant cells. Thus, the Golgi and TGN are probably functionally distinct organelles in plants.

A BAR-Domain Protein SH3P2, Which Binds to Phosphatidylinositol 3-Phosphate and ATG8, Regulates Autophagosome Formation in Arabidopsis

This work identifies SH3P2 as a novel regulator of autophagy and provides a conserved model for autophagosome biogenesis in Arabidopsis. Autophagosome-related structures, such as isolation membranes and ER-derived omegasome-like structures, are characterized and SH3P2 is shown to bind to PI3P and regulate autophagosome formation via the association with the PI3K complex and ATG8. Autophagy is a well-defined catabolic mechanism whereby cytoplasmic materials are engulfed into a structure termed the autophagosome. In plants, little is known about the underlying mechanism of autophagosome formation. In this study, we report that SH3 DOMAIN-CONTAINING PROTEIN2 (SH3P2), a Bin-Amphiphysin-Rvs domain–containing protein, translocates to the phagophore assembly site/preautophagosome structure (PAS) upon autophagy induction and actively participates in the membrane deformation process. Using the SH3P2–green fluorescent protein fusion as a reporter, we found that the PAS develops from a cup-shaped isolation membranes or endoplasmic reticulum–derived omegasome-like structures. Using an inducible RNA interference (RNAi) approach, we show that RNAi knockdown of SH3P2 is developmentally lethal and significantly suppresses autophagosome formation. An in vitro membrane/lipid binding assay demonstrates that SH3P2 is a membrane-associated protein that binds to phosphatidylinositol 3-phosphate. SH3P2 may facilitate membrane expansion or maturation in coordination with the phosphatidylinositol 3-kinase (PI3K) complex during autophagy, as SH3P2 promotes PI3K foci formation, while PI3K inhibitor treatment inhibits SH3P2 from translocating to autophagosomes. Further interaction analysis shows that SH3P2 associates with the PI3K complex and interacts with ATG8s in Arabidopsis thaliana, whereby SH3P2 may mediate autophagy. Thus, our study has identified SH3P2 as a novel regulator of autophagy and provided a conserved model for autophagosome biogenesis in Arabidopsis.

Multiple cytosolic and transmembrane determinants are required for the trafficking of SCAMP1 via an ER―Golgi―TGN―PM pathway

How polytopic plasma membrane (PM) proteins reach their destination in plant cells remains elusive. Using transgenic tobacco BY-2 cells, we previously showed that the rice secretory carrier membrane protein 1 (SCAMP1), an integral membrane protein with four transmembrane domains (TMDs), is localized to the PM and trans-Golgi network (TGN). Here, we study the transport pathway and sorting signals of SCAMP1 by following its transient expression in tobacco BY-2 protoplasts and show that SCAMP1 reaches the PM via an endoplasmic reticulum (ER)-Golgi-TGN-PM pathway. Loss-of-function and gain-of-function analysis of various green fluorescent protein (GFP) fusions with SCAMP1 mutations further demonstrates that: (i) the cytosolic N-terminus of SCAMP1 contains an ER export signal; (ii) the transmembrane domain 2 (TMD2) and TMD3 of SCAMP1 are essential for Golgi export; (iii) SCAMP1 TMD1 is essential for TGN-to-PM targeting; (iv) the predicted topology of SCAMP1 and its various mutants remain identical as demonstrated by protease protection assay. Therefore, both the cytosolic N-terminus and TMD sequences of SCAMP1 play integral roles in mediating its transport to the PM via an ER-Golgi-TGN pathway.

SCAMPs Highlight the Developing Cell Plate during Cytokinesis in Tobacco BY-2 Cells

We previously demonstrated that rice (Oryza sativa) SECRETORY CARRIER MEMBRANE PROTEIN1 (OsSCAMP1)-yellow fluorescent protein in transgenic tobacco (Nicotiana tabacum) Bright Yellow-2 cells locates to the plasma membrane and to motile punctate structures, which represent the trans-Golgi network/early endosome and are tubular-vesicular in nature. Here, we now show that SCAMPs are diverted to the cell plate during cytokinesis dividing Bright Yellow-2 cells. As cells progress from metaphase to cytokinesis, punctate OsSCAMP1-labeled structures begin to collect in the future division plane. Together with the internalized endosomal marker FM4-64, they then become incorporated into the cell plate as it forms and expands. This was confirmed by immunogold electron microscopy. We also monitored for the Golgi apparatus and the prevacuolar compartment (PVC)/multivesicular body. Golgi stacks tend to accumulate in the vicinity of the division plane, but the signals are clearly separate to the cell plate. The situation with the PVC (labeled by green fluorescent protein-BP-80) is not so clear. Punctate BP-80 signals are seen at the advancing periphery of the cell plate, which was confirmed by immunogold electron microscopy. Specific but weak labeling was observed in the cell plate, but no evidence for a fusion of the PVC/multivesicular body with the cell plate could be obtained. Our data, therefore, support the notion that cell plate formation is mainly a secretory process involving mass incorporation of domains of the trans-Golgi network/early endosome membrane. We regard the involvement of multivesicular late endosomes in this process to be equivocal.

The role of the endoplasmic reticulum in peroxisome biogenesis.

Peroxisomes are essential cellular organelles involved in lipid metabolism. Patients affected by severe peroxisome biogenesis disorders rarely survive their first year. Genetic screens in several model organisms have identified more than 30 PEX genes that are required for the formation of functional peroxisomes. Despite significant work on the PEX genes, the biogenic origin of peroxisomes remains controversial. For at least two decades, the prevailing model postulated that peroxisomes propagate by growth and fission of preexisting peroxisomes. In this review, we focus on the recent evidence supporting a new, semiautonomous model of peroxisomal biogenesis. According to this model, peroxisomal membrane proteins (PMPs) traffic from the endoplasmic reticulum (ER) to the peroxisome by a vesicular budding, targeting, and fusion process while peroxisomal matrix proteins are imported into the organelle by an autonomous, posttranslational mechanism. We highlight the contradictory conclusions reached to answer the question of how PMPs are inserted into the ER. We then review what we know and what still remains to be elucidated about the mechanism of PMP exit from the ER and the contribution of preperoxisomal vesicles to mature peroxisomes. Finally, we discuss discrepancies in our understanding of de novo peroxisome biogenesis in wild-type cells. We anticipate that resolving these key issues will lead to a more complete picture of peroxisome biogenesis.

Vacuolar Degradation of Two Integral Plasma Membrane Proteins, AtLRR84A and OsSCAMP1, Is Cargo Ubiquitination‐Independent and Prevacuolar Compartment‐Mediated in Plant Cells

In plant cells, how integral plasma membrane (PM) proteins are degraded in a cargo ubiquitination‐independent manner remains elusive. Here, we studied the degradative pathway of two plant PM proteins: AtLRR84A, a type I integral membrane protein belonging to the leucine‐rich repeat receptor‐like kinase protein family, and OsSCAMP1 (rice secretory carrier membrane protein 1), a tetraspan transmembrane protein located on the PM and trans‐Golgi network (TGN) or early endosome (EE). Using wortmannin and ARA7(Q69L) mutant that could enlarge the multivesicular body (MVB) or prevacuolar compartment (PVC) as tools, we demonstrated that, when expressed as green fluorescent protein (GFP) fusions in tobacco BY‐2 or Arabidopsis protoplasts, both AtLRR84A and OsSCAMP1 were degraded in the lytic vacuole via the internal vesicles of MVB/PVC in a cargo ubiquitination‐independent manner. Such MVB/PVC‐mediated vacuolar degradation of PM proteins was further supported by immunocytochemical electron microscopy (immunoEM) study showing the labeling of the fusions on the internal vesicles of the PVC/MVB. Thus, cargo ubiquitination‐independent and PVC‐mediated degradation of PM proteins in the vacuole is functionally operated in plant cells.

Unique COPII component AtSar1a/AtSec23a pair is required for the distinct function of protein ER export in Arabidopsis thaliana

Significance The underlying mechanisms causing the functional diversity of small GTPase Sar1 paralogs in coat protein complex II-mediated protein endoplasmic reticulum (ER) export remain elusive in higher organisms. Arabidopsis contains five Sar1 homologs. In this study, we show that AtSar1a exhibits a distinct localization and effects on ER cargo export in plants through a unique interaction with the COPII coat protein AtSec23a. This specific pairing is required for the distinct function in ER export under ER stress in Arabidopsis. Our results point to a mechanism underlying the functional diversity of COPII paralogs in eukaryotes. Secretory proteins traffic from endoplasmic reticulum (ER) to Golgi via the coat protein complex II (COPII) vesicle, which consists of five cytosolic components (Sar1, Sec23-24, and Sec13-31). In eukaryotes, COPII transport has diversified due to gene duplication, creating multiple COPII paralogs. Evidence has accumulated, revealing the functional heterogeneity of COPII paralogs in protein ER export. Sar1B, the small GTPase of COPII machinery, seems to be specialized for large cargo secretion in mammals. Arabidopsis contains five Sar1 and seven Sec23 homologs, and AtSar1a was previously shown to exhibit different effects on α-amylase secretion. However, mechanisms underlying the functional diversity of Sar1 paralogs remain unclear in higher organisms. Here, we show that the Arabidopsis Sar1 homolog AtSar1a exhibits distinct localization in plant cells. Transgenic Arabidopsis plants expressing dominant-negative AtSar1a exhibit distinct effects on ER cargo export. Mutagenesis analysis identified a single amino acid, Cys84, as being responsible for the functional diversity of AtSar1a. Structure homology modeling and interaction studies revealed that Cys84 is crucial for the specific interaction of AtSar1a with AtSec23a, a distinct Arabidopsis Sec23 homolog. Structure modeling and coimmunoprecipitation further identified a corresponding amino acid, Cys484, on AtSec23a as being essential for the specific pair formation. At the cellular level, the Cys484 mutation affects the distinct function of AtSec23a on vacuolar cargo trafficking. Additionally, dominant-negative AtSar1a affects the ER export of the transcription factor bZIP28 under ER stress. We have demonstrated a unique plant pair of COPII machinery function in ER export and the mechanism underlying the functional diversity of COPII paralogs in eukaryotes.