The yeast GTP-binding YPT1 protein and a mammalian counterpart are associated with the secretion machinery

Segev, Nava; Mulholland, Jon; Botstein, David

doi:10.1016/0092-8674(88)90433-3

Cited by 685 publications

(480 citation statements)

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“…Considering that Secl2p is recycling between the ER and the cis Golgi in the wild-type yeast cells and that Rerlp is involved in proper localization of Secl2p, it would be reasonable to assume the location of the Rerlp function to be either in the ER or in the Golgi. The punctate staining pattern of strongly suggests the latter possibility; a number of Golgi proteins such as Yptlp, Sec7p, and Kex2p show punctate localization in the cytoplasm (Segev et al, 1988;Franzusoff et al, 1991;Nishikawa and Nakano, 1991;Redding et al, 1991; also see below). To support this possibility, the effect of the sec7 mutation was examined.…”

Section: Rerm Encodes a Hydrophobic Proteinmentioning

confidence: 92%

“…Immunofluorescence of Yptlp gives a punctate pattern that is exaggerated in the sec7 mutant like the staining of Rerl-3HAp (Segev et al, 1988). More importantly, a study by immunoelectron microscopy has shown that Yptlp is in fact associated with the Golgi membranes (Preuss et al, 1992).…”

Section: Rer1mentioning

confidence: 92%

“…Because the antibody against Ochlp could not detect any signal by immunofluorescence, we chose the anti-Yptlp antibody for double staining experiments. Yptlp is a small GTPase required for targeting and fusion of ER-derived vesicles with the Golgi apparatus (Segev et al, 1988;Segev, 1991).…”

Section: Rerm Encodes a Hydrophobic Proteinmentioning

confidence: 99%

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Membrane protein retrieval from the Golgi apparatus to the endoplasmic reticulum (ER): characterization of the RER1 gene product as a component involved in ER localization of Sec12p.

Sato

Nishikawa

Nakano

1995

MBoC

101

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Yeast Sec12p, a type II transmembrane glycoprotein, is required for formation of transport vesicles from the endoplasmic reticulum (ER). Biochemical and morphological analyses have suggested that Sec12p is localized to the ER by two mechanisms: static retention in the ER and dynamic retrieval from the early region of the Golgi apparatus. The rer1 mutant we isolated in a previous study mislocalizes the authentic Sec12p to the later compartments of the Golgi. To understand the role of RER1 on Sec12p localization, we cloned the gene and determined its reading frame. RER1 encodes a hydrophobic protein of 188 amino acid residues containing four putative membrane spanning domains. The rer1 null mutant is viable. Even in the rer1 disrupted cells, immunofluorescence of Sec12p stains the ER, implying that the retention system is still operating in the mutant. To determine the subcellular localization of Rer1p, an epitope derived from the influenza hemagglutinin was added to the C-terminus of Rer1p and the cells expressing this tagged but functional protein were observed by immunofluorescence microscopy. The anti-HA monoclonal antibody stains the cells in a punctate pattern that is typical for Golgi proteins and clearly distinct from the ER staining. This punctate staining was in fact exaggerated in the sec7 mutant that accumulates the Golgi membranes at the restrictive temperature. Furthermore, double staining of Rer1p and Ypt1p, a GTPase that is known to reside in the Golgi apparatus, showed good colocalization. Subcellular fractionation experiments indicated that the fractionation pattern of Rer1p was similar to that of an early Golgi protein, Och1p. From these results, we suggest that Rer1p functions in the Golgi membrane to return Sec12p that has escaped from the static retention system of the ER.

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Section: Rerm Encodes a Hydrophobic Proteinmentioning

confidence: 92%

Section: Rer1mentioning

confidence: 92%

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Membrane protein retrieval from the Golgi apparatus to the endoplasmic reticulum (ER): characterization of the RER1 gene product as a component involved in ER localization of Sec12p.

Sato

Nishikawa

Nakano

1995

MBoC

101

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“…The YPT1 (a small GTPase involved in secretion [96]) and PEX8 (a peroxisomal matrix protein [65]) promoters have not been widely used, probably because of their low expression levels, since the goal of the majority of researchers using the P. pastoris expression system is to obtain maximum amounts of expressed foreign proteins [18]. The remainder of this section will discuss the more recent developments regarding P. pastoris promoters.…”

Section: The Pichia Expression Systemsmentioning

confidence: 99%

Heterologous protein production using the Pichia pastoris expression system

Macauley-Patrick

Fazenda

McNeil

et al. 2005

Yeast

1,097

633

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The Pichia pastoris expression system is being used successfully for the production of various recombinant heterologous proteins. Recent developments with respect to the Pichia expression system have had an impact on not only the expression levels that can be achieved, but also the bioactivity of various heterologous proteins. We review here some of these recent developments, as well as strategies for reducing proteolytic degradation of the expressed recombinant protein at cultivation, cellular and protein levels. The problems associated with post-translational modifications performed on recombinant proteins by P. pastoris are discussed, including the effects on bioactivity and function of these proteins, and some engineering strategies for minimizing unwanted glycosylations. We pay particular attention to the importance of optimizing the physicochemical environment for efficient and maximal recombinant protein production in bioreactors and the role of process control in optimizing protein production is reviewed. Finally, future aspects of the use of the P. pastoris expression system are discussed with regard to the production of complex membrane proteins, such as G protein-coupled receptors, and the industrial and clinical importance of these proteins.

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“…More is known about the role of proteins of the YPT1/SEC4/rab family, which we will refer to as rab proteins. Several rab proteins have been shown to be required at specific steps of membrane transport, both in yeast (Goud et al, 1988;Segev et al, 1988) and in mammalian cells Plutner et al, 1991;Bucci et al, 1992;Lombardi et al, 1993). Moreover, every member of that family that has been localized exhibits a specific subcellular distribution (Goud and McCaffrey, 1991;Gruenberg and Clague, 1992).…”

Section: Introductionmentioning

confidence: 99%

The N-terminal domain of a rab protein is involved in membrane-membrane recognition and/or fusion.

et al. 1994

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Proteins of the YPT1/SEC4/rab family are well documented to be involved in the regulation of membrane transport. We have previously reported that rab5 regulates endosome-endosome recognition and/or fusion in vitro. Here, we show that this process depends on the rab5 N-terminal domain. Treatment of early endosomal membranes at a low trypsin concentration essentially abolished fusion and cleaved rab5 to a 1 kDa smaller polypeptide. Two-dimensional gel analysis suggested that rab5 is one of the few, if not the only, polypeptides cleaved by trypsin under these conditions. Whereas endosome fusion could be stimulated by cytosol prepared from cells overexpressing rab5 (and thus containing high amounts of the protein), this stimulation was abolished by trypsin-treatment of the cytosol. Trypsin-treated cytosol prepared from mock-transfected cells, which contains very low amounts of rab5, showed no inhibitory activity indicating that rab5 is the target of trypsin in these experiments. Purified rab5 prepared after expression in Escherichia coli was treated with trypsin, which cleaved the protein at the N-terminus. A synthetic peptide of rab5 N-terminal domain inhibited endosome fusion in our cell-free assay. A version of the same peptide truncated at the N-terminus or a peptide of rab3 N-terminal domain were without effects. Altogether, these observations suggest that the N-terminal domain of rab5 is involved in the process of early endosome recognition and/or fusion, presumably because it interacts with another component of the transport machinery.

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The yeast GTP-binding YPT1 protein and a mammalian counterpart are associated with the secretion machinery

Cited by 685 publications

References 32 publications

Membrane protein retrieval from the Golgi apparatus to the endoplasmic reticulum (ER): characterization of the RER1 gene product as a component involved in ER localization of Sec12p.

Membrane protein retrieval from the Golgi apparatus to the endoplasmic reticulum (ER): characterization of the RER1 gene product as a component involved in ER localization of Sec12p.

Heterologous protein production using the Pichia pastoris expression system

The N-terminal domain of a rab protein is involved in membrane-membrane recognition and/or fusion.

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