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Chris Hawes

Publications and source records attributed to Chris Hawes.

At least 19 recordsLinked to original sources
PubMed2006

Plant N-glycan processing enzymes employ different targeting mechanisms for their spatial arrangement along the secretory pathway.

The processing of N-linked oligosaccharides in the secretory pathway requires the sequential action of a number of glycosidases and glycosyltransferases. We studied the spatial distribution of several type II membrane-bound enzymes from Glycine max, Arabidopsis thaliana, and Nicotiana tabacum. Glucosidase I (GCSI) localized to the endoplasmic reticulum (ER), alpha-1,2 mannosidase I (ManI) and N-acetylglucosaminyltransferase I (GNTI) both targeted to the ER and Golgi, and beta-1,2 xylosyltransferase localized exclusively to Golgi stacks, corresponding to the order of expected function. ManI deletion constructs revealed that the ManI transmembrane domain (TMD) contains all necessary targeting information. Likewise, GNTI truncations showed that this could apply to other type II enzymes. A green fluorescent protein chimera with ManI TMD, lengthened by duplicating its last seven amino acids, localized exclusively to the Golgi and colocalized with a trans-Golgi marker (ST52-mRFP), suggesting roles for protein-lipid interactions in ManI targeting. However, the TMD lengths of other plant glycosylation enzymes indicate that this mechanism cannot apply to all enzymes in the pathway. In fact, removal of the first 11 amino acids of the GCSI cytoplasmic tail resulted in relocalization from the ER to the Golgi, suggesting a targeting mechanism relying on protein-protein interactions. We conclude that the localization of N-glycan processing enzymes corresponds to an assembly line in the early secretory pathway and depends on both TMD length and signals in the cytoplasmic tail.

Arabidopsis↗
PubMed2006

The plant ER-Golgi interface: a highly structured and dynamic membrane complex.

As compared with other eukaryotic cells, plants have developed an endoplasmic reticulum (ER)-Golgi interface with very specific structural characteristics. ER to Golgi and Golgi to ER transport appear not to be dependent on the cytoskeleton, and ER export sites have been found closely associated with Golgi bodies to constitute entire mobile units. However, the molecular machinery involved in membrane trafficking seems to be relatively conserved among eukaryotes. Therefore, a challenge for plant scientists is to determine how these molecular machineries work in a different structural and dynamic organization. This review will focus on some aspects of membrane dynamics that involve coat proteins, SNAREs (soluble N-ethylmaleimide-sensitive factor attachment receptor proteins), lipids, and lipid-interacting proteins.

Endoplasmic Reticulum↗
PubMed2006

Mapping the Arabidopsis organelle proteome.

A challenging task in the study of the secretory pathway is the identification and localization of new proteins to increase our understanding of the functions of different organelles. Previous proteomic studies of the endomembrane system have been hindered by contaminating proteins, making it impossible to assign proteins to organelles. Here we have used the localization of organelle proteins by the isotope tagging technique in conjunction with isotope tags for relative and absolute quantitation and 2D liquid chromatography for the simultaneous assignment of proteins to multiple subcellular compartments. With this approach, the density gradient distributions of 689 proteins from Arabidopsis thaliana were determined, enabling confident and simultaneous localization of 527 proteins to the endoplasmic reticulum, Golgi apparatus, vacuolar membrane, plasma membrane, or mitochondria and plastids. This parallel analysis of endomembrane components has enabled protein steady-state distributions to be determined. Consequently, genuine organelle residents have been distinguished from contaminating proteins and proteins in transit through the secretory pathway.

Arabidopsis↗
PubMed2006

In tobacco leaf epidermal cells, the integrity of protein export from the endoplasmic reticulum and of ER export sites depends on active COPI machinery.

Trafficking of secretory proteins between the endoplasmic reticulum (ER) and the Golgi apparatus depends on coat protein complexes I (COPI) and II (COPII) machineries. To date, full characterization of the distribution and dynamics of these machineries in plant cells remains elusive. Furthermore, except for a presumed linkage between COPI and COPII for the maintenance of ER protein export, the mechanisms by which COPI influences COPII-mediated protein transport from the ER in plant cells are largely uncharacterized. Here we dissect the dynamics of COPI in intact cells using live-cell imaging and fluorescence recovery after photobleaching analyses to provide insights into the distribution of COPI and COPII machineries and the mechanisms by which COPI influences COPII-mediated protein export from the ER. We found that Arf1 and coatomer are dynamically associated with the Golgi apparatus and that the COPII coat proteins Sec24 and Sec23 localize at ER export sites that track with the Golgi apparatus in tobacco leaf epidermal cells. Arf1 is also localized at additional structures that originate from the Golgi apparatus but that lack coatomer, supporting the model that Arf1 also has a coatomer-independent role for post-Golgi protein transport in plants. When ER to Golgi protein transport is inhibited by mutations that hamper Arf1-GTPase activity without directly disrupting the COPII machinery for ER protein export, Golgi markers are localized in the ER and the punctate distribution of Sec24 and Sec23 at the ER export sites is lost. These findings suggest that Golgi membrane protein distribution is maintained by the balanced action of COPI and COPII systems, and that Arf1-coatomer is most likely indirectly required for forward trafficking out of the ER due to its role in recycling components that are essential for differentiation of the ER export domains formed by the Sar1-COPII system.

ADP-Ribosylation Factor 1↗
PubMed2005

Sec22 and Memb11 are v-SNAREs of the anterograde endoplasmic reticulum-Golgi pathway in tobacco leaf epidermal cells.

Distinct sets of soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs) are distributed to specific intracellular compartments and catalyze membrane fusion events. Although the central role of these proteins in membrane fusion is established in nonplant systems, little is known about their role in the early secretory pathway of plant cells. Analysis of the Arabidopsis (Arabidopsis thaliana) genome reveals 54 genes encoding SNARE proteins, some of which are expected to be key regulators of membrane trafficking between the endoplasmic reticulum (ER) and the Golgi. To gain insights on the role of SNAREs of the early secretory pathway in plant cells, we have cloned the Arabidopsis v-SNAREs Sec22, Memb11, Bet11, and the t-SNARE Sed5, and analyzed their distribution in plant cells in vivo. By means of live cell imaging, we have determined that these SNAREs localize at the Golgi apparatus. In addition, Sec22 was also distributed at the ER. We have then focused on understanding the function of Sec22 and Memb11 in comparison to the other SNAREs. Overexpression of the v-SNAREs Sec22 and Memb11 but not of the other SNAREs induced collapse of Golgi membrane proteins into the ER, and the secretion of a soluble secretory marker was abrogated by all SNAREs. Our studies suggest that Sec22 and Memb11 are involved in anterograde protein trafficking at the ER-Golgi interface.

Biomarkers↗
PubMed2005

Photoactivation of GFP reveals protein dynamics within the endoplasmic reticulum membrane.

Components of the plant cell secretory pathway, including the endoplasmic reticulum and Golgi apparatus, are in constant motion. The photoactivation of GFP has been used to determine that proteins within the membrane of the ER flow as the ER is remodelled. Measurement of the rate at which activated GFP moves away from the activation spot shows that this motion is much faster than would be expected if membrane components moved simply by diffusion. Treatment with latrunculin to depolymerize the actin cytoskeleton stops ER remodelling and reduces the rate of GFP movement to that expected from diffusion alone. This suggests that myosin binds directly or indirectly to ER membrane proteins and actively moves them around over the actin scaffold. Tracking of Golgi body movement was used to demonstrate that they move at the same rate and in the same direction as do photoactivated ER surface proteins. Golgi bodies, therefore, move with, and not over, the surface of the ER. These observations support the current theory of continuity between Golgi bodies and discrete ER exit sites in the ER membrane.

Bridged Bicyclo Compounds, Heterocyclic↗
PubMed2005

Holding it all together? Candidate proteins for the plant Golgi matrix.

A combination of electron microscopy and fluorescence microscopy has provided us with a global picture of the structure of the plant Golgi apparatus. However, the components that shape this structure remain elusive. In other organisms, members of the golgin family of coiled-coil proteins are essential for Golgi structure and organisation. Putative Arabidopsis and rice homologues of some golgin family members can be identified using database searches. Likewise, the heterogeneous group of multi-subunit-tethering complexes is responsible for crucial transport steps that affect Golgi structure and cisternal organisation in animals and yeasts. The Arabidopsis genome harbours possible homologues for the majority of the subunits of these complexes, suggesting that they also operate in the plant kingdom.

Golgi Apparatus↗
PubMed2005

AtPEX2 and AtPEX10 are targeted to peroxisomes independently of known endoplasmic reticulum trafficking routes.

Controversy exists in the literature over the involvement of the endoplasmic reticulum (ER) in the delivery of membrane proteins to peroxisomes. In this study, the involvement of the ER in the trafficking of two Arabidopsis (Arabidopsis thaliana) peroxisomal membrane proteins was investigated using confocal laser scanning microscopy of living cells expressing fusions between enhanced yellow fluorescent protein (eYFP) and AtPEX2 and AtPEX10. The fusion proteins were always detected in peroxisomes and cytosol irrespective of the location of the eYFP tag or the level of expression. The cytosolic fluorescence was not due to cleavage of the eYFP reporter from the C-terminal fusion proteins. Blocking known ER transport routes using the fungal metabolite Brefeldin A or expressing dominant negative mutants of Sar1 or RabD2a had no effect on the trafficking of AtPEX2 and AtPEX10 to peroxisomes. We conclude that AtPEX2 and AtPEX10 are inserted into peroxisome membranes directly from the cytosol.

Arabidopsis↗
PubMed2005

The plant Golgi apparatus--going with the flow.

The plant Golgi apparatus is composed of many separate stacks of cisternae which are often associated with the endoplasmic reticulum and which in many cell types are motile. In this review, we discuss the latest data on the molecular regulation of Golgi function. The concept of the Golgi as a distinct organelle is challenged and the possibility of a continuum between the endoplasmic reticulum and Golgi is proposed.

COP-Coated Vesicles↗
PubMed2005

The plant Golgi apparatus--going with the flow.

The plant Golgi apparatus is composed of many separate stacks of cisternae which are often associated with the endoplasmic reticulum and which in many cell types are motile. In this review, we discuss the latest data on the molecular regulation of Golgi function. The concept of the Golgi as a distinct organelle is challenged and the possibility of a continuum between the endoplasmic reticulum and Golgi is proposed.

COP-Coated Vesicles↗
PubMed2005

An Arabidopsis GRIP domain protein locates to the trans-Golgi and binds the small GTPase ARL1.

GRIP domain proteins are a class of golgins that have been described in yeast and animals. They locate to the trans-Golgi network and are thought to play a role in endosome-to-Golgi trafficking. The Arabidopsis GRIP domain protein, AtGRIP, fused to the green fluorescent protein (GFP), locates to Golgi stacks but does not exactly co-locate with the Golgi marker sialyl transferase (ST)-mRFP, nor with the t-SNAREs Memb11, SYP31 and BS14a. We conclude that the location of AtGRIP is further to the trans side of the stack than STtmd-mRFP. The 185-aa C-terminus of AtGRIP containing the GRIP domain targeted GFP to the Golgi, although a proportion of the fusion protein was still found in the cytosol. Mutation of a conserved tyrosine (Y717) to alanine in the GRIP domain disrupted Golgi localization. ARL1 is a small GTPase required for Golgi targeting of GRIP domain proteins in other systems. An Arabidopsis ARL1 homologue was isolated and shown to target to Golgi stacks. The GDP-restricted mutant of ARL1, AtARL1-T31N, was observed to locate partially to the cytosol, whereas the GTP-restricted mutant AtARL1-Q71L labelled the Golgi and a population of small structures. Increasing the levels of AtARL1 in epidermal cells increased the proportion of GRIP-GFP fusion protein on Golgi stacks. We show, moreover, that AtARL1 interacted with the GRIP domain in a GTP-dependent manner in vitro in affinity chromatography and in the yeast two-hybrid system. This indicates that AtGRIP and AtARL1 interact directly. We conclude that the pathway involving ARL1 and GRIP domain golgins is conserved in plants.

Amino Acid Sequence↗
PubMed2005

Cell biology of the plant Golgi apparatus.

The higher plant Golgi apparatus, comprising many individual stacks of membrane bounded cisternae, is one of the most enigmatic of the cytoplasmic organelles. Not only can the stacks receive material from the endoplasmic reticulum, process it and target it to the correct cellular destination, but they can also synthesise and export complex carbohydrates and lipids and most likely act as one end point of the endocytic pathway. In many cells such processing and sorting can take place while the stacks are moving within the cytoplasm and, remarkably, the organelle manages to retain its structural integrity. This review considers some of the latest data and views on transport both to and from the Golgi and the mechanisms by which such activity is regulated.

Biological Transport, Active↗
PubMed2004

AtRabF2b (Ara7) acts on the vacuolar trafficking pathway in tobacco leaf epidermal cells.

Rab GTPases are universal key regulators of intracellular secretory trafficking events. In particular, Rab 5 homologues have been implicated in endocytic events and in the vacuolar pathway. In this study, we investigate the location and function of a member of this family, AtRabF2b (Ara7) in tobacco (Nicotiana tabacum) leaf epidermal cells using a live cell imaging approach. Fluorescent-tagged AtRabF2b[wt] localized to the prevacuolar compartment and Golgi apparatus, as determined by coexpression studies with fluorescent markers for these compartments. Mutations that impair AtRabF2b function also alter the subcellular location of the GTPase. In addition, coexpression studies of the protein with the vacuole-targeted aleurain-green fluorescent protein (GFP) and rescue experiments with wild-type AtRabF2b indicate that the dominant-negative mutant of AtRabF2b causes the vacuolar marker to be secreted to the apoplast. Our results indicate a clear role of AtRabF2b in the vacuolar trafficking pathway.

Biomarkers↗
PubMed2004

Endoplasmic reticulum export sites and Golgi bodies behave as single mobile secretory units in plant cells.

In contrast with animals, plant cells contain multiple mobile Golgi stacks distributed over the entire cytoplasm. However, the distribution and dynamics of protein export sites on the plant endoplasmic reticulum (ER) surface have yet to be characterized. A widely accepted model for ER-to-Golgi transport is based on the sequential action of COPII and COPI coat complexes. The COPII complex assembles by the ordered recruitment of cytosolic components on the ER membrane. Here, we have visualized two early components of the COPII machinery, the small GTPase Sar1p and its GTP exchanging factor Sec12p in live tobacco (Nicotiana tabacum) leaf epidermal cells. By in vivo confocal laser scanning microscopy and fluorescence recovery after photobleaching experiments, we show that Sar1p cycles on mobile punctate structures that track with the Golgi bodies in close proximity but contain regions that are physically separated from the Golgi bodies. By contrast, Sec12p is uniformly distributed along the ER network and does not accumulate in these structures, consistent with the fact that Sec12p does not become part of a COPII vesicle. We propose that punctate accumulation of Sar1p represents ER export sites (ERES). The sites may represent a combination of Sar1p-coated ER membranes, nascent COPII membranes, and COPII vectors in transit, which have yet to lose their coats. ERES can be induced by overproducing Golgi membrane proteins but not soluble bulk-flow cargos. Few punctate Sar1p loci were observed that are independent of Golgi bodies, and these may be nascent ERES. The vast majority of ERES form secretory units that move along the surface of the ER together with the Golgi bodies, but movement does not influence the rate of cargo transport between these two organelles. Moreover, we could demonstrate using the drug brefeldin A that formation of ERES is strictly dependent on a functional retrograde transport route from the Golgi apparatus.

Agrobacterium tumefaciens↗
PubMed2004

A GFP-based assay reveals a role for RHD3 in transport between the endoplasmic reticulum and Golgi apparatus.

We describe the use of a secreted form of Aequoria victoria green fluorescent protein (secGFP) in a non-invasive live cell assay of membrane traffic in Arabidopsis thaliana. We show that in comparison to GFP-HDEL, which accumulates in the endoplasmic reticulum (ER), secGFP generates a weak fluorescence signal when transported to the apoplast. The fluorescence of secGFP in the apoplast can be increased by growth of seedlings on culture medium buffered at pH 8.1, suggesting that apoplastic pH is responsible, at least in part, for the low fluorescence intensity of seedlings expressing secGFP. Inhibition of secGFP transport between the ER and plasma membrane (PM), either by Brefeldin A (BFA) treatment or by genetic intervention results in increased intracellular secGFP accumulation accompanied by an increase in the secGFP fluorescence intensity. secGFP thus provides a valuable tool for forward and reverse genetic analysis of membrane traffic and endomembrane organisation in Arabidopsis. Using this assay for quantitative sublethal perturbation of secGFP transport, we identify a role for root hair defective 3 (RHD3) in transport of secreted and Golgi markers between the ER and the Golgi apparatus.

Arabidopsis↗
PubMed2004

Transport of ricin and 2S albumin precursors to the storage vacuoles of Ricinus communis endosperm involves the Golgi and VSR-like receptors.

We have studied the transport of proricin and pro2S albumin to the protein storage vacuoles of developing castor bean (Ricinus communis L.) endosperm. Immunoelectron microscopy and cell fractionation reveal that both proteins travel through the Golgi apparatus and co-localize throughout their route to the storage vacuole. En route to the PSV, the proteins co-localize in large (>200 nm) vesicles, which are likely to represent developing storage vacuoles. We further show that the sequence-specific vacuolar sorting signals of both proricin and pro2SA bind in vitro to proteins that have high sequence similarity to members of the VSR/AtELP/BP-80 vacuolar sorting receptor family, generally associated with clathrin-mediated traffic to the lytic vacuole. The implications of these findings in relation to the current model for protein sorting to storage vacuoles are discussed.

2S Albumins, Plant↗
PubMed2003

An Arabidopsis pex10 null mutant is embryo lethal, implicating peroxisomes in an essential role during plant embryogenesis.

Peroxisomes participate in many important functions in plants, including seed reserve mobilization, photorespiration, defense against oxidative stress, and auxin and jasmonate signaling. In mammals, defects in peroxisome biogenesis result in multiple system abnormalities, severe developmental delay, and death, whereas in unicellular yeasts, peroxisomes are dispensable unless required for growth of specific substrates. PEX10 encodes an integral membrane protein required for peroxisome biogenesis in mammals and yeast. To investigate the importance of PEX10 in plants, we characterized a Ds insertion mutant in the PEX10 gene of Arabidopsis (AtPEX10). Heterozygous AtPEX10::dissociation element mutants show normal vegetative phenotypes under optimal growth conditions, but produce about 20% abnormal seeds. The embryos in the abnormal seeds are predominantly homozygous for the disruption allele. They show retarded development and some morphological abnormalities. No viable homozygous mutant plants were obtained. AtPEX10 fused to yellow fluorescent protein colocalized with green fluorescent protein-serine-lysine-leucine, a well-documented peroxisomal marker, suggesting that AtPEX10 encodes a peroxisomal protein that is essential for normal embryo development and viability.

Amino Acid Sequence↗