Characterization of the unprocessed and processed forms of rab6 expressed in baculovirus/insect cell systems

Yang, Chenzhi; Mayau, Véronique; Godeau, François; Goud, Bruno

doi:10.1016/0006-291x(92)91903-4

Cited by 13 publications

(13 citation statements)

References 25 publications

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“…After washings, cells were recultured in Grace's medium supplemented with 1 mM methionine and 1 mM cysteine for various time periods. Cells were then lysed directly in Triton X-114 and subjected to phase separation as previously described [48]. Aqueous and detergents phases were then immunoprecipitated using affinitypuFified anti-rab6p [6].…”

Section: Pulse-chase Experimentsmentioning

confidence: 99%

“…We have previously shown that two major forms of rab6p can be obtained when it is expressed in the baculovirus/insect cell system; a protein which has a molecular mass of 24 kDa as estimated by SDSPAGE and which is found in the cytosol and a 23-kDa protein found mainly associated with the membrane of insect cells [48]. The 24-kDa protein partitions into the aqueous phase of Triton X-114 (hydrophilic) whereas the 23-kDa protein partitions in the detergent phase (hydrophobic; [48] and Figs 1 and 2B).…”

Section: Purification Of Cytosolic (Unprocessed) and Membrane-bound (mentioning

confidence: 99%

“…The 24-kDa protein partitions into the aqueous phase of Triton X-114 (hydrophilic) whereas the 23-kDa protein partitions in the detergent phase (hydrophobic; [48] and Figs 1 and 2B). This latter form is isoprenylated [48] and carboxymethylated (Mayau, V. and Goud, B., unpublished results). The pulse-chase experiment shown in Fig.…”

Section: Purification Of Cytosolic (Unprocessed) and Membrane-bound (mentioning

confidence: 99%

“…During the course of purification of cytosolic rab6p, we have sometimes noted the appearance of a protein migrating at approximately 21 kDa on SDSPAGE. This protein is recognized by antibodies raised against rab6p and binds to GTP on immunoblots [48]. It probably corresponds to a degradation product of rab6p.…”

Section: Purification Of Cytosolic (Unprocessed) and Membrane-bound (mentioning

confidence: 99%

“…These proteins are not post-translationally processed, but it is generally assumed that they display the same properties as the mature, lipid-modified forms found in the membranes of mammalian cells. Processed proteins can be obtained when the genes are expressed in the baculovirusl insect cell system and several small CiTP-binding proteins have been recently purified from infected insect cells [46, 471. We have previously shown that, when expressed in insect cells, two major forms of rab6p are obtained; a protein found in the cytosol of infected insect cells which represents the unprocessed precursor form of rab6p and a membranebound isoprenylated protein [48]. In this study, we have punfied to homogeneity the two forms of rab6p.…”

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confidence: 91%

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Comparison of the biochemical properties of unprocessed and processed forms of the small GTP‐binding protein, rab6p

Yang

Mollat

Chaffotte

et al. 1993

European Journal of Biochemistry

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The rab6 protein (rab6p) belongs to a large family of ras-like low-molecular-mass GTP-binding proteins thought to be involved in the regulation of intracellular transport in mammalian cells. When expressed in the baculovirus/insect cell system, two major forms of rab6p are obtained; a 24-kDa cytosolic unprocessed form and a 23-kDa membrane-bound form which represents the processed lipid-modified protein. Here, we have purified both forms to homogeneity and we have studied and compared their biochemical properties. Unprocessed and processed rab6p display similar bindingrate constants (k,,") for GDP and GTP (2-1.9 pM-' min-'). However, significant differences exist in the dissociation constants of bound guanine nucleotides. Processed rab6p in low and high magnesium solutions displays similar koff values for GTP and GDP. However, unprocessed rab6p has a ken, value higher for GDP than for GTP in both low and high magnesium solutions. Their intrinsic GTPase activities also differ ; unprocessed rab6p has an almost undetectable GTPase activity, whereas that of processed rab6p is in the same range as that reported for other ras and ras-like GTPbinding proteins (0.012 % 0.002 min-'). These results suggest that post-translational modifications of rab6p might induce subtle changes in the three-dimensional structure of the protein which affect the guanine-nucleotide-binding/hydrolysis activity.

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Section: Pulse-chase Experimentsmentioning

confidence: 99%

Section: Purification Of Cytosolic (Unprocessed) and Membrane-bound (mentioning

confidence: 99%

Section: Purification Of Cytosolic (Unprocessed) and Membrane-bound (mentioning

confidence: 99%

Section: Purification Of Cytosolic (Unprocessed) and Membrane-bound (mentioning

confidence: 99%

mentioning

confidence: 91%

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Comparison of the biochemical properties of unprocessed and processed forms of the small GTP‐binding protein, rab6p

Yang

Mollat

Chaffotte

et al. 1993

European Journal of Biochemistry

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Characterization and subcellular localization of a small GTP-binding protein (Ara-4) fromArabidopsis: conditional expression under control of the promoter of the gene for heat-shock protein HSP81-1

et al. 1996

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Small GTP-binding proteins belonging to the rab/YPT family play key roles at various steps in intracellular transport pathways in yeast and mammalian cells. Many members of rab/YPT family have been isolated from plants to date. However, detailed information about the localization and function of the gene products remains limited, even though intracellular transport is likely to be involved in important phenomena such as cell elongation, transport of storage proteins, determination and maintenance of cell polarity and intercellular signal transduction. We have attempted to establish transgenic Arabidopsis plants that overexpress ARA-4, a rab/YPT homologue in order to analyze the function and the localization of the gene product. For overexpression and also for regulation of the expression of this gene, the promoter of the gene for HSP81-1 was employed to drive the transcription of ARA-4 in transgenic plants. The response of the introduced genes to heat shock was analyzed. Upon heat-shock treatment, the ARA-4 gene was efficiently transcribed and translated. The induction of ARA-4 by heat shock was transient, and at least two distinct forms of this protein were found in membrane and cytosolic fractions from transgenic plants. Prolonged incubation after heat shock reduced the amount of the cytosolic form of the induced protein, and the cytosolic form of the protein thus probably represents the unprocessed precursor. Using transgenic plants, we determined the subcellular localization of the product of ARA-4. The protein was predominantly localized on Golgi-derived vesicles, Golgi cisternae and the trans-Golgi network.

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The role of small G-proteins in the transport of newly synthesized rhodopsin

Deretic¹

1995

Progress in Retinal and Eye Research

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Characterization of the unprocessed and processed forms of rab6 expressed in baculovirus/insect cell systems

Cited by 13 publications

References 25 publications

Comparison of the biochemical properties of unprocessed and processed forms of the small GTP‐binding protein, rab6p

Comparison of the biochemical properties of unprocessed and processed forms of the small GTP‐binding protein, rab6p

Characterization and subcellular localization of a small GTP-binding protein (Ara-4) fromArabidopsis: conditional expression under control of the promoter of the gene for heat-shock protein HSP81-1

The role of small G-proteins in the transport of newly synthesized rhodopsin

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