Farnesylation of Pex19p is not essential for peroxisome biogenesis in yeast and mammalian cells

Vastiau, Ilse; Anthonio, Erin; Brams, Marijke; Brees, Chantal; Young, Stephen G.; Velde, Sam Van de; Wanders, Ronald J. A.; Mannaerts, Guy P.; Baes, Myriam; Veldhoven, Paul P. Van; Fransen, Marc

doi:10.1007/s00018-006-6110-y

Cited by 27 publications

(20 citation statements)

References 72 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Our recombinant Pex19p was made in E. coli; thus it lacks the C-terminal prenylation group attached to the native protein in yeast (11,27). Nonetheless, the recombinant proteins, as well as a GST-Pex19p chimera, were functional in our cell-free budding reaction.…”

Section: Discussionmentioning

confidence: 99%

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

Lam¹,

Yoda²,

Schekman

2010

Proc. Natl. Acad. Sci. U.S.A.

117

128

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionmentioning

confidence: 99%

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

Lam¹,

Yoda²,

Schekman

2010

Proc. Natl. Acad. Sci. U.S.A.

117

128

View full text Add to dashboard Cite

show abstract

“…The plasmids Dsred-PTS1, bicistronic Pex19 / EGFP-PTS1, Pex19, EGFP-Pex19, Pex13p-EGFP, and pCMV-TIRAP-flag were described (Fransen et al, 2001; Horng et al, 2001; Vastiau et al, 2006). Pex19 was amplified from EGFP-Pex19 and inserted into the retroviral vector pMSCV IRES GFP.…”

Section: Methodsmentioning

confidence: 99%

Peroxisomes Are Signaling Platforms for Antiviral Innate Immunity

Dixit

Boulant

Zhang

et al. 2010

Cell

724

697

View full text Add to dashboard Cite

“…The pGEM4-Pex5(C11S)p has been previously described (24). The monocistronic mammalian expression plasmid encoding a EGFP fusion protein containing at its C terminus a peroxisomal targeting signal type 1 (EGFP-PTS1) has been described elsewhere (33). The bicistronic mammalian expression vectors coding for EGFP-PTS1 and the nontagged long isoforms of Pex5p, Pex5(C11S)p, or Pex5(C11K)p were constructed by amplifying the corresponding cDNAs by PCR (primers, 5Ј-GGGAGATCTACCATGGCAATGCGGGAGCTG-3Ј and 5Ј-GCCCGTCGACCTGTCACTGGGGCAGGCCAAAC-3Ј) and cloning the BglII/SalI-digested PCR products into the multiple cloning site of pIRES-EGFP-PTS1 (34) digested with the same restriction enzymes.…”

Section: Methodsmentioning

confidence: 99%

Properties of the Ubiquitin-Pex5p Thiol Ester Conjugate

Grou

Carvalho

Pinto

et al. 2009

Journal of Biological Chemistry

111

View full text Add to dashboard Cite

Pex5p, the peroxisomal protein cycling receptor, binds newly synthesized peroxisomal matrix proteins in the cytosol and promotes their translocation across the organelle membrane. During its transient passage through the membrane, Pex5p is monoubiquitinated at a conserved cysteine residue, a requisite for its subsequent ATP-dependent export back into the cytosol. Here we describe the properties of the soluble and membrane-bound monoubiquitinated Pex5p species (Ub-Pex5p). Our data suggest that 1) Ub-Pex5p is deubiquitinated by a combination of context-dependent enzymatic and nonenzymatic mechanisms; 2) soluble Ub-Pex5p retains the capacity to interact with the peroxisomal import machinery in a cargo-dependent manner; and 3) substitution of the conserved cysteine residue of Pex5p by a lysine results in a quite functional protein both in vitro and in vivo. Additionally, we show that MG132, a proteasome inhibitor, blocks the import of a peroxisomal reporter protein in vivo.Since the discovery of the ubiquitin-conjugating cascade nearly 30 years ago, thousands of proteins have been shown to be modified by ubiquitin (1, 2). In many cases ubiquitination of a protein is linked to its proteasomal degradation (3), whereas in a growing number of examples, ubiquitination of a protein is used as a transient modification to modulate its biological properties (for a review see Ref. 4). Regardless of the final outcome, it is generally assumed and in many cases demonstrated that ubiquitin is covalently attached through an amide bond involving the carboxyl group of the last glycine of ubiquitin on one hand, and an amino group of the targeted protein on the other (5). Recent findings from several laboratories, however, suggest that this rule is not always valid, and proteins ubiquitinated at serines and threonines (yielding oxyesters) or even cysteines (forming thiol esters) have been identified (6 -10).Protein ubiquitination at cysteine residues is a particularly puzzling phenomenon for two reasons. First, on a thermodynamic basis it is the least favorable event (the approximate free energy changes for acyl shifts from a thiol ester to a thiol, alcohol, and amine are 0, Ϫ2.4, and Ϫ11 kcal/mol, respectively (11, 12)). Second, although data on the half-lives of ubiquitin-protein thiol ester conjugates under physiologically relevant conditions are scarce, it is known that ubiquitin thiol esters are easily disrupted by nucleophiles such as GSH (13), raising the possibility that, to some degree, proteins subjected to this kind of conjugation may undergo futile ubiquitination/deubiquitination cycles. Thus, a thiol ester bond appears not to be the most efficient way to link ubiquitin to a protein, unless, of course, the aim is to create an activated (easily transferable) form of ubiquitin, as is in fact the case with ubiquitin-activating enzymes (E1s), 4 ubiquitin-conjugating enzymes (E2s), and some ubiquitin ligases (E3s) (2).In the last years we have been characterizing Pex5p, one of the three presently known proteins claimed to be ub...

show abstract

Farnesylation of Pex19p is not essential for peroxisome biogenesis in yeast and mammalian cells

Cited by 27 publications

References 72 publications

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

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

Peroxisomes Are Signaling Platforms for Antiviral Innate Immunity

Properties of the Ubiquitin-Pex5p Thiol Ester Conjugate

Contact Info

Product

Resources

About