Retrograde transport of mutant ricin to the endoplasmic reticulum with subsequent translocation to cytosol

Rapak, Andrzej; Falnes, Pål Ø.; Olsnes, Sjur

doi:10.1073/pnas.94.8.3783

Cited by 220 publications

(261 citation statements)

References 56 publications

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“…the C-terminus of the A chain was modified by the addition of overlapping tyrosine sulphation and N-glycosylation sites [10]. It was found that only ricin A chain that had been both tyrosinesulphated (an event occurring in the TGN) and core N-glycosylated (an event occurring in the ER) was translocated to the cytosol, implying that passage through both the TGN and ER was necessary for entry into the cytosol and inhibition of protein synthesis.…”

Section: Introductionmentioning

confidence: 99%

Cell polarization is required for ricin sensitivity in a Caco-2 cell line selected for ricin resistance

Jackman

Ellis

Gray

et al. 1999

Biochemical Journal

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It has been proposed that killing of mammalian cells by ricin requires efficient endocytic delivery to the trans-Golgi network (TGN) prior to retrograde transport to the endoplasmic reticulum and entry to the cytosol. In polarized epithelial cells, an efficient membrane-traffic pathway to the TGN is present from the basolateral but not the apical plasma-membrane domain. Thus one can hypothesize that a ricin-resistant phenotype might be demonstrated by polarized cells that fail to differentiate and thus fail to develop an efficient membrane-traffic pathway from the basolateral plasma membrane to the TGN. We have isolated and studied a ricin-resistant Caco-2 cell clone (Caco-2-RCAr clone 2) which, when grown on plastic, was deficient in differentiation, measured by the development of polarized-cell-surface marker enzymes. The deficiency in differentiation was partially reversed, and ricin sensitivity was restored, when the cells were grown on filter supports. Our data provide the first evidence of a ricin-resistant cell line where resistance is due to the lack of development of polarized cell surfaces. The observed ricin resistance is consistent with the requirement that ricin is delivered to the TGN before its A chain enters the cytosol to mediate cell killing.

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Section: Introductionmentioning

confidence: 99%

Cell polarization is required for ricin sensitivity in a Caco-2 cell line selected for ricin resistance

Jackman

Ellis

Gray

et al. 1999

Biochemical Journal

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“…SLO was used to selectively permeabilize cell plasma membranes to allow cytosol collection (Rapak et al, 1997). First, we ensured that SLO permeabilized the plasma membrane but not the ER membrane.…”

Section: Cytosolic Translocation Of [ 125 I]vtamentioning

confidence: 99%

Membrane cytosolic translocation of verotoxin A1 subunit in target cells

Tam

Lingwood

2007

Microbiology

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“…In that case, a mechanism for uptake and delivery of Avr9 to the ER or additional components for an indirect interaction with Cf-9 must be postulated. Interestingly, several toxins have been found to be transported all the way from the cell surface, through endosomes and the Golgi apparatus, to the ER: the Shiga toxin (Sandvig et al, 1992), the cholera toxin (Sandvig et al, 1996), and ricin (Rapak et al, 1997). Avr9 binding studies, using purified PM, showed that tomato plants exhibited a high-affinity binding site for Avr9 whether or not they carried Cf-9 (Kooman-Gersmann et al, 1996).…”

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confidence: 99%

The C-Terminal Dilysine Motif Confers Endoplasmic Reticulum Localization to Type I Membrane Proteins in Plants

2000

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The tomato Cf-9 disease resistance gene encodes a type I membrane protein carrying a cytosolic dilysine motif. In mammals and yeast, this motif promotes the retrieval of type I membrane proteins from the Golgi apparatus to the endoplasmic reticulum (ER). To test whether the C-terminal KKXX signal of Cf-9 is functional as a retrieval motif and to investigate its role in plants, green fluorescent protein (GFP) was fused to the transmembrane domain of Cf-9 and expressed in yeast, Arabidopsis, and tobacco cells. The fusion protein was targeted to the ER in each of these expression systems, and mutation of the KKXX motif to NNXX led to secretion of the fusion protein. In yeast, the mutant protein reached the vacuole, but plants secreted it as a soluble protein after proteolytic removal of the transmembrane domain. Triple hemagglutinin (HA)-tagged full-length Cf-9 was also targeted to the ER in tobacco cells, and cleavage was also observed for the NNXX mutant protein, suggesting an endoprotease recognition site located within the Cf-9 lumenal sequence common to both the GFP-and the HA-tagged fusions. Our results indicate that the KKXX motif confers ER localization in plants as well as mammals and yeast and that Cf-9 is a resident protein of the ER. INTRODUCTIONProteins resident in the endoplasmic reticulum (ER) contain structural motifs responsible for their correct subcellular localization. Many soluble ER proteins, such as BiP or protein disulfide isomerase, carry a C-terminal tetrapeptide H/KDEL motif for localization in the ER (Munro and Pelham, 1987;Pelham et al., 1988;Mazzarella et al., 1990;Andres et al., 1991; Denecke et al., 1992 Denecke et al., , 1993Napier et al., 1992). A membrane-bound receptor encoded by the yeast ERD2 gene retrieves escaping HDEL-marked proteins from the Golgi apparatus or salvage compartment back to the ER Semenza et al., 1990). Homologs of the Erd2 receptor have been cloned from mammals Pelham, 1990, 1992; Tang et al., 1993) and plants ( Lee et al., 1993), highlighting conservation of the H/KDEL motif and retrieval mechanism.In mammals and yeast, the cytosolic dilysine motif is critical for ER localization of type I membrane proteins (Nilsson et al., 1989;Jackson et al., 1990). The two lysine residues need to be in either the Ϫ 3, Ϫ 4 (KKXX) or Ϫ 3, Ϫ 5 (KXKXX) positions relative to the C terminus, and no other basic amino acid can be substituted (Jackson et al., 1990(Jackson et al., , 1993. Mutation of lysine residues leads to expression of the reporter protein on the cell surface in mammals (Nilsson et al., 1989;Jackson et al., 1990) and to its vacuolar delivery in yeast (Gaynor et al., 1994). Studies of the post-translational modification kinetics of dilysine-tagged reporter proteins have demonstrated a constant cycling of the reporter from the ER to the Golgi apparatus and back to the ER (Jackson et al., 1993;Gaynor et al., 1994). The retrieval mechanism is mediated by vesicular Golgi apparatus-to-ER retrograde transport, involving the coat protein I (COPI) complex (Cosson and...

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Retrograde transport of mutant ricin to the endoplasmic reticulum with subsequent translocation to cytosol

Cited by 220 publications

References 56 publications

Cell polarization is required for ricin sensitivity in a Caco-2 cell line selected for ricin resistance

Cell polarization is required for ricin sensitivity in a Caco-2 cell line selected for ricin resistance

Membrane cytosolic translocation of verotoxin A1 subunit in target cells

The C-Terminal Dilysine Motif Confers Endoplasmic Reticulum Localization to Type I Membrane Proteins in Plants

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