Amino- and carboxy-terminal domains of the yeast Rab escort protein are both required for binding of Ypt small G proteins.

Bauer, Brigitte; Lorenzetti, Stefano; Miączyńska, Marta; Bui, Duc Minh; Schweyen, Rudolf J.; Ragnini-Wilson, Antonella

doi:10.1091/mbc.7.10.1521

Cited by 21 publications

(51 citation statements)

References 48 publications

(58 reference statements)

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“…The media YPD, YPGal, SD, and SGal (supplemented with amino acids and bases as required) were previously described (20). Yeast transformations were performed according to Ref.…”

Section: Methodsmentioning

confidence: 99%

“…The resulting construct, named M6GDI, was recloned in the XbaI-KpnI sites of the YEplac181 vector (plasmid YEpM6GDI). The plasmids YEpM6PN, YEp351, YEpM6⌬Nhe, and YEpM6⌬Xba were previously described (7,20). Plasmid amplification and propagation were performed as described previously (20).…”

Section: Methodsmentioning

confidence: 99%

“…Absence of the endogenous Mrs6p was determined by immunoblotting analyses as described previously (20). For Mrs6p overexpression, the strain AR4-6A was transformed with YEpM6PN (7), precultured in ϪUra SD medium for 48 h, diluted to A 600 ϭ 0.2 in YPD, and grown at 30°C for 8 h.…”

Section: Methodsmentioning

confidence: 99%

“…Subcellular Fractionation-Protein extracts were prepared from the wild type strain AR4-6A by glass bead lysis as described (20) and subsequently centrifuged at 100,000 ϫ g for 1 h to obtain the total membrane fraction (P100) and cytosol (S100). The membrane fraction was washed once with lysis buffer and resuspended in 10 mM Tris-Cl, pH 7.4.…”

Section: Methodsmentioning

confidence: 99%

“…Mrs6 and Ypt1 Protein Expression, Antibodies, Immunodetection, and Geranylgeranylation Assay-Mrs6p and Ypt1p were expressed as His 6 -tagged proteins in Escherichia coli as described previously (20). The anti-Mrs6 polyclonal antibody SLM6 -1 (20) was used in this study.…”

Section: Methodsmentioning

confidence: 99%

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The Yeast Rab Escort Protein Binds Intracellular Membranes in Vivo and in Vitro

Miączyńska

Lorenzetti

Bialek

et al. 1997

Journal of Biological Chemistry

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In both mammals and yeast, intracellular vesicular transport depends on the correct shuttling between membrane and cytosol of the Rab/Ypt small G proteins. Membrane association of these proteins requires prenylation by the Rab geranylgeranyl transferase that recognizes a complex formed by the Rab/Ypt protein and the Rab escort protein (REP). After prenylation the Rab/ Ypt protein is delivered to the target membranes by REP. Little is known about the early steps of the Rab-REP complex formation and where this association occurs in the cell. Although prenylation is believed to take place in the cytosol, we show that the yeast Rab escort protein Mrs6 is present in both soluble and particulate fractions of cell extracts. Mrs6p is associated with the heavy microsomal fraction that contains endoplasmic reticulum-Golgi membranes but is absent in the plasma membrane, vacuoles, mitochondria, and microsomal subfraction associated with mitochondria. The solubilization pattern of the particulate pool of Mrs6p implies that this protein is peripherally but tightly associated with membranes via hydrophobic interactions and metal ions. We also report that the C terminus of Mrs6p is important for maintaining the solubility of the protein because its deletion or replacement with the C terminus of RabGDI results in a protein that localizes only to membranes.

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“…The media YPD, YPGal, SD, and SGal (supplemented with amino acids and bases as required) were previously described (20). Yeast transformations were performed according to Ref.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

The Yeast Rab Escort Protein Binds Intracellular Membranes in Vivo and in Vitro

Miączyńska

Lorenzetti

Bialek

et al. 1997

Journal of Biological Chemistry

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DNA sequencing and analysis of a 67·4 kb region from the right arm ofSchizosaccharomyces pombe chromosome II reveals 28 open reading frames including the geneshis5,pol5,ppa2,rip1,rpb8 andskb1

Xiang

Lyne

Wood

et al. 1999

Yeast

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67 393 bp of contiguous DNA located between markers cdc18 and cdc14 on the right arm of fission yeast chromosome II has been sequenced as part of the European Union Schizosaccharomyces pombe genome sequencing project. The complete sequence, contained in cosmid clones c15C4 and c21H7, has been determined on both strands. Sequence analysis shows that it contains 28 open reading frames capable of coding for proteins, 16 split by one or more introns, but no tRNA, rRNA or transposon sequences. The gene density is one per 2·4 kb. Six genes have been previously described (his5, pol5, ppa2, rip1, rpb8 and skb1) and 22 are novel. Of the novel genes, 14 have significant similarity with proteins of known function, three have similarities with proteins of unknown function and five show no extensive similarities with known proteins. Sequence similarities suggest that three of the novel genes encode ATP‐dependent RNA helicases, two encode transcription factor components and others encode a G‐protein, a dehydrogenase, a Rab escort protein, an Abc1‐like protein, a lipase, an ATP‐binding transport protein, an amino acid permease, an acid phosphatase and a mannosyltransferase. The sequence has been submitted to the EMBL database under entries: SPBC15C4 (Accession No. AL023290), SPBC21H7 (AL023286), SPBC14C8 (part)(AL022305) and SPBC16H5 (part)(AL022104). Copyright © 1999 John Wiley & Sons, Ltd.

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Ypt protein prenylation depends on the interplay among levels of Rab escort protein and geranylgeranyl diphosphate in yeast cells

Miączyńska

Wagner

Bauer

et al. 2001

Yeast

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Farnesyl diphosphate (FPP), an intermediate of the sterol biosynthetic pathway, is used by farnesyl transferase to farnesylate, among others, the Ras proteins, and by geranylgeranyl diphosphate synthase to produce geranylgeranyl diphosphate (GGPP). GGPP is then transferred by geranylgeranyl transferase II (GGTase II) to Rab/Ypt members of the Ras superfamily known to be required at all stages of vesicle transport in both mammals and yeast. Formation of a complex between a Rab/Ypt protein and an accessory protein named the Rab escort protein (REP) is a prerequisite for GGTase II substrate recognition. Little is known about the factors that regulate GGTase II activity in living cells but, based on available data, it seems possible that vesicle transport in higher eukaryotes is regulated by the levels of prenylated Rab/Ypt proteins in the cells. Here we show that the levels of REP play an important role in regulating GGTase II activity in yeast cells if sufficient substrates are present. Moreover, overexpression of REP causes, directly or indirectly, an increased level of Ypt substrates available for prenylation, which in turn leads to the depletion of the GGPP pool in the cell. Overall our data suggest that the levels of REP and the availability of GGPP play a role in regulating Ypt protein prenylation.

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Amino- and carboxy-terminal domains of the yeast Rab escort protein are both required for binding of Ypt small G proteins.

Abstract: The Rab escort protein (REP) is

Cited by 21 publications

References 48 publications

The Yeast Rab Escort Protein Binds Intracellular Membranes in Vivo and in Vitro

The Yeast Rab Escort Protein Binds Intracellular Membranes in Vivo and in Vitro

DNA sequencing and analysis of a 67·4 kb region from the right arm ofSchizosaccharomyces pombe chromosome II reveals 28 open reading frames including the geneshis5,pol5,ppa2,rip1,rpb8 andskb1

Ypt protein prenylation depends on the interplay among levels of Rab escort protein and geranylgeranyl diphosphate in yeast cells

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