Aldo Ceriotti

Protein Quality Control Mechanisms and Protein Storage in the Endoplasmic Reticulum. A Conflict of Interests

More than 30 years ago it was observed that the storage proteins of maize ( Zea mays ) seeds accumulate as “bulges or localized dilatations along the endoplasmic reticulum cisternae” of developing endosperm cells ([Khoo and Wolf, 1970][1]). It later became evident that, whereas the widespread

Ricin A chain without its partner B chain is degraded after retrotranslocation from the endoplasmic reticulum to the cytosol in plant cells.

When expressed in tobacco cells, the catalytic subunit of the dimeric ribosome inactivating protein, ricin, is first inserted into the endoplasmic reticulum (ER) and then degraded in a manner that can be partially inhibited by the proteasome inhibitor clasto-lactacystin β-lactone. Consistent with the implication of cytosolic proteasomes, degradation of ricin A chain is brefeldin A-insensitive and the polypeptides that accumulate in the presence of the proteasome inhibitor are not processed in a vacuole-specific fashion. Rather, these stabilized polypeptides are in part deglycosylated by a peptide:N-glycanase-like activity. Taken together, these results indicate that ricin A chain, albeit a structurally native protein, can behave as a substrate for ER to cytosol export, deglycosylation in the cytosol, and proteasomal degradation. Furthermore, retrotranslocation of this protein is not tightly coupled to proteasomal activity. These data are consistent with the hypothesis that ricin A chain can exploit the ER-associated protein degradation pathway to reach the cytosol. Although well characterized in mammalian and yeast cells, the operation of a similar pathway to the cytosol of plant cells has not previously been demonstrated.

Wild emmer genome architecture and diversity elucidate wheat evolution and domestication

Genomics and domestication of wheat Modern wheat, which underlies the diet of many across the globe, has a long history of selection and crosses among different species. Avni et al. used the Hi-C method of genome confirmation capture to assemble and annotate the wild allotetraploid wheat (Triticum turgidum). They then identified the putative causal mutations in genes controlling shattering (a key domestication trait among cereal crops). They also performed an exome capture–based analysis of domestication among wild and domesticated genotypes of emmer wheat. The findings present a compelling overview of the emmer wheat genome and its usefulness in an agricultural context for understanding traits in modern bread wheat. Science, this issue p. 93 A polyploid wheat genome assembly elucidates wheat domestication history. Wheat (Triticum spp.) is one of the founder crops that likely drove the Neolithic transition to sedentary agrarian societies in the Fertile Crescent more than 10,000 years ago. Identifying genetic modifications underlying wheat’s domestication requires knowledge about the genome of its allo-tetraploid progenitor, wild emmer (T. turgidum ssp. dicoccoides). We report a 10.1-gigabase assembly of the 14 chromosomes of wild tetraploid wheat, as well as analyses of gene content, genome architecture, and genetic diversity. With this fully assembled polyploid wheat genome, we identified the causal mutations in Brittle Rachis 1 (TtBtr1) genes controlling shattering, a key domestication trait. A study of genomic diversity among wild and domesticated accessions revealed genomic regions bearing the signature of selection under domestication. This reference assembly will serve as a resource for accelerating the genome-assisted improvement of modern wheat varieties.

Saporin and ricin A chain follow different intracellular routes to enter the cytosol of intoxicated cells

Several protein toxins, such as the potent plant toxin ricin, enter mammalian cells by endocytosis and undergo retrograde transport via the Golgi complex to reach the endoplasmic reticulum (ER). In this compartment the catalytic moieties exploit the ER‐associated degradation (ERAD) pathway to reach their cytosolic targets. Bacterial toxins such as cholera toxin or Pseudomonas exotoxin A carry KDEL or KDEL‐like C‐terminal tetrapeptides for efficient delivery to the ER. Chimeric toxins containing monomeric plant ribosome‐inactivating proteins linked to various targeting moieties are highly cytotoxic, but it remains unclear how these molecules travel within the target cell to reach cytosolic ribosomes. We investigated the intracellular pathways of saporin, a monomeric plant ribosome‐inactivating protein that can enter cells by receptor‐mediated endocytosis. Saporin toxicity was not affected by treatment with Brefeldin A or chloroquine, indicating that this toxin follows a Golgi‐independent pathway to the cytosol and does not require a low pH for membrane translocation. In intoxicated Vero or HeLa cells, ricin but not saporin could be clearly visualized in the Golgi complex using immunofluorescence. The saporin signal was not evident in the Golgi, but was found to partially overlap with that of a late endosome/lysosome marker. Consistently, the toxicities of saporin or saporin‐based targeted chimeric polypeptides were not enhanced by the addition of ER retrieval sequences. Thus, the intracellular movement of saporin differs from that followed by ricin and other protein toxins that rely on Golgi‐mediated retrograde transport to reach their retrotranslocation site.

The Binding Protein Associates with Monomeric Phaseolin

The association of the binding protein (BiP) with newly synthesized proteins in the endoplasmic reticulum (ER) of developing bean (Phaseolus vulgaris) cotyledonary cells was investigated. ATP-sensitive association with many polypeptides was detected. The fraction of newly synthesized polypeptides associated with BiP varies among different proteins. The relationship between subunit assembly and binding to BiP was investigated in the case of the vacuolar trimeric glycoprotein phaseolin. In spite of the presence of a significant pool of phaseolin trimers in the ER, only monomeric phaseolin is found in association with BiP. On the whole, our results point to a general role of BiP in the synthesis of plant secretory proteins and indicate that, in the case of phaseolin, BiP binding sites are concealed during structural maturation in the ER, either before or upon formation of trimers. Our results also indicate that trimerization does not constitute a rate-limiting step in the transport of phaseolin to the protein storage vacuoles.

Endoplasmic reticulum-associated degradation of ricin A chain has unique and plant-specific features.

Proteins that fail to fold in the endoplasmic reticulum (ER) or cannot find a pattern for assembly are often disposed of by a process named ER-associated degradation (ERAD), which involves transport of the substrate protein across the ER membrane (dislocation) followed by rapid proteasome-mediated proteolysis. Different ERAD substrates have been shown to be ubiquitinated during or soon after dislocation, and an active ubiquitination machinery has been found to be required for the dislocation of certain defective proteins. We have previously shown that, when expressed in tobacco (Nicotiana tabacum) protoplasts, the A chain of the heterodimeric toxin ricin is degraded by a pathway that closely resembles ERAD but is characterized by an unusual uncoupling between the dislocation and the degradation steps. Since lysine (Lys) residues are a major target for ubiquitination, we have investigated the effects of changing the Lys content on the retrotranslocation and degradation of ricin A chain in tobacco protoplasts. Here we show that modulating the number of Lys residues does not affect recognition events within the ER lumen nor the transport of the protein from this compartment to the cytosol. Rather, the introduced modifications have a clear impact on the degradation of the dislocated protein. While the substitution of the two Lys residues present in ricin A chain with arginine slowed down degradation, the introduction of four extra lysyl residues had an opposite effect and converted the ricin A chain to a standard ERAD substrate that is disposed via a process in which dislocation and degradation steps are tightly coupled.

The Rate of Phaseolin Assembly Is Controlled by the Glucosylation State of Its N-Linked Oligosaccharide Chains.

Many of the proteins that are translocated into the endoplasmic reticulum are glycosylated with the addition of a 14-saccharide core unit (Glc3Man9GlcNAc2) to specific asparagine residues of the nascent polypeptide. Glucose residues are then removed by endoplasmic reticulum-located glucosidases, with diglucosylated and monoglucosylated intermediates being formed. In this study, we used a cell-free system constituted of wheat germ extract and bean microsomes to examine the role of glucose trimming in the structural maturation of phaseolin, a trimeric glycoprotein that accumulates in the protein storage vacuoles of bean seeds. Removal of glucose residues from the N-linked chains of phaseolin was blocked by the glucosidase inhibitors castanospermine and N-methyldeoxynojirimycin. If glucose trimming was not allowed to occur, the assembly of phaseolin was accelerated. Conversely, polypeptides bearing partially trimmed glycans were unable to form trimers. The effect of castanospermine on the rate of assembly was much more pronounced for phaseolin polypeptides that have two glycans but was also evident when a single glycan chain was present, indicating that glycan clustering can modulate the effect of glucose trimming on the rate of trimer formation. Therefore, the position of glycan chains and their accessibility to the action of glucosidases can be fundamental elements in the control of the structural maturation of plant glycoproteins.

Exogenous Protein Expression in Xenopus Oocytes

The oocytes of the South African clawed frog Xenopus laevis have been widely used as a reliable system for the expression and characterization of different types of proteins, including ion channels and membrane receptors. The large size and resilience of these oocytes make them easy to handle and to microinject with different molecules such as natural mRNAs, cRNAs, and antibodies. A variety of methods can then be used to monitor the expression of the proteins encoded by the microinjected mRNA/cRNA, and to perform a functional characterization of the heterologous polypeptides. In this chapter, after describing the equipment required to maintain X. laevis in the laboratory and to set up a microinjection system, we provide detailed procedures for oocyte isolation, micropipet and cRNA preparation, and oocyte microinjection. A method for the labeling of oocyte-synthesized proteins and for the immunological detection of the heterologous polypeptides is also described.

ER Dislocation: Cdc48p/p97 Gets Into the AAAct

Misfolded or unassembled proteins present in the lumen of the endoplasmic reticulum are exported to the cytosol and degraded. Recent studies have implicated a complex containing the AAA ATPase Cdc48p/p97 in the export process.

The position of the proricin vacuolar targeting signal is functionally important

Ricin is synthesised as an ER-targeted precursor containing an enzymatic A chain and a galactose-binding B chain separated by a 12-amino acid linker propeptide. This internal propeptide is known to contain a sequence-specific vacuolar sorting signal whose functionality depends on the presence of an isoleucine residue. Conversion of this isoleucine to glycine completely abolished vacuolar targeting of proricin and led to its secretion. However, when this mutated signal was positioned at the C-terminus of a normally secreted reporter, vacuolar targeting of a significant fraction still occurred. Likewise, when the corrupted linker was C-terminally exposed within its natural context following the mature ricin A chain, and then co-expressed with ricin B chain, toxin heterodimers were still partially transported to tobacco cell vacuoles. By contrast, when placed at the N-terminus of the secreted reporter, or at the N-terminus of ricin B chain for co-expression with ricin A chain, the propeptide behaved most strikingly as a sequence-specific vacuolar targeting signal that, when mutated, resulted in complete secretion of the proteins. It would appear that the position of the linker peptide influences the specificity of its vacuolar targeting function.