The green fluorescent protein targets secretory proteins to the yeast vacuole

Kunze, Irene; Hensel, Goetz; Adler, Klaus; Bernard, Jeffrey R.; Neubohn, Birgit; Nilsson, Cecilia; Stoltenburg, Regina; Kohlwein, Sepp D.; Kunze, Gotthard

doi:10.1016/s0005-2728(99)00006-7

Cited by 53 publications

(44 citation statements)

References 47 publications

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“…Some Sec-GFP was clearly found in the cell fraction (c) both from a wild-type and a mutant strain ( ⌬ vps10), but the amount of GFP was significantly lower when the gene for Vps10p was disrupted. This finding indicated, as described previously (Kunze et al, 1999), that GFP is sent to the vacuole in wild-type yeast, and our results additionally demonstrated that this is due to Vps10p activity. This is also in accord with previous observations showing a low specificity for Vps10p that can send to the vacuole for degradation many proteins that are either foreign to yeast or not properly folded (Hong et al, 1996).…”

supporting

confidence: 92%

“…It has been reported that a potentially secreted form of GFP was sent to the vacuole in wild-type yeast (Kunze et al, 1999). To determine whether this was also the case in a strain with a disrupted gene for the yeast vacuolar receptor Vps10p ( ⌬ vps10), we transformed this mutant yeast with the two control constructs represented in Figure 1, a secreted GFP (Sec-GFP) and a GFP fused to the yeast CPY vacuolar signal (CPY-GFP).…”

Section: Gfp Is An Appropriate Reporter In Which To Study Plant Vsds mentioning

confidence: 99%

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Demonstration in Yeast of the Function of BP-80, a Putative Plant Vacuolar Sorting Receptor

et al. 2001

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BP-80, later renamed VSR PS-1 , is a putative receptor involved in sorting proteins such as proaleurain to the lytic vacuole, with its N-terminal domain recognizing the vacuolar sorting determinant. Although all VSR PS-1 characteristics and in vitro binding properties described so far favored its receptor function, this function remained to be demonstrated. Here, we used green fluorescent protein (GFP) as a reporter in a yeast mutant strain defective for its own vacuolar receptor, Vps10p. By expressing VSR PS-1 together with GFP fused to the vacuolar sorting determinant of petunia proaleurain, we were able to efficiently redirect the reporter to the yeast vacuole. VSR PS-1 is ineffective on GFP either alone or when fused with another type of plant vacuolar sorting determinant from a chitinase. The plant VSR PS-1 therefore interacts specifically with the proaleurain vacuolar sorting determinant in vivo, and this interaction leads to the transport of the reporter protein through the yeast secretory pathway to the vacuole. This finding demonstrates VSR PS-1 receptor function but also emphasizes the differences in the spectrum of ligands between Vps10p and its plant equivalent. INTRODUCTIONThe plant secretory system is far less easy to study than its yeast counterpart, mainly due to the lack of an equivalent mutant collection. Plant and yeast cells, in opposition to mammalian cells, do not use the mannose 6-phosphatebased lysosomal sorting tag (Kornfeld, 1992); instead, the sorting information is carried by a peptidic sequence. In yeast, the two vacuolar proteins carboxypeptidase Y (CPY; Johnson et al., 1987) and proteinase A (Klionsky and Emr, 1989) both are produced with an N-terminal propeptide responsible for the vacuolar location of the mature protein. Almost 50 mutants defective for vacuolar protein sorting ( vps ) have allowed the identification of proteins involved in the yeast vacuolar sorting pathway. One of these proteins, Vps10p, plays a central role because it is the vacuolar receptor for CPY (Marcusson et al., 1994). Vps10p also is able to send foreign or malfolded proteins to the vacuole for degradation in a process that is believed to be independent of any sorting signal (Hong et al., 1996).In plants, two main types of vacuolar sorting determinants (VSDs) have been well studied and are believed to correspond to two separate vacuolar sorting pathways. The first type could be defined as a sequence-specific VSD (ssVSD) and has been well studied for barley aleurain (Holwerda et al., 1992) and sweet potato sporamin A (Matsuoka and Nakamura, 1991). For these two examples, the VSD is located within an N-terminal propeptide and contains a conserved tetrapeptide NPIR, the Ile being essential (Matsuoka and Nakamura, 1999). The position of this category of VSD seems to be less important than its sequence specificity, as shown with sporamin A (Koide et al., 1997). The second type of VSD is found in C-terminal propeptides (Ct-VSD); it is rather short with no obvious sequence conservation but needs to be accessibl...

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supporting

confidence: 92%

Section: Gfp Is An Appropriate Reporter In Which To Study Plant Vsds mentioning

confidence: 99%

Demonstration in Yeast of the Function of BP-80, a Putative Plant Vacuolar Sorting Receptor

et al. 2001

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“…3a) revealed that EGFP was mainly located in the vacuole (stained using FM4-64; Vida & Emr, 1995) in these cells when using the native EpxL-RT leader or MFa. Both vacuolar and cytosolic location has been previously reported by other groups for MFa (Kottmeier et al, 2011;Kunze et al, 1999).…”

Section: Secretion Of Recombinant Proteins By Testing the Exp1 Nativesupporting

confidence: 73%

Multistep processing of the secretion leader of the extracellular protein Epx1 in Pichia pastoris and implications for protein localization

et al. 2015

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Secretion leaders are required to direct nascent proteins to the secretory pathway. They are of interest in the study of intracellular protein transport, and are required for the production of secretory recombinant proteins. Secretion leaders are processed in two steps in the endoplasmic reticulum and Golgi. Although yeast cells typically contain about 150 proteins entering the secretory pathway, only a low number of proteins are actually secreted to the cell supernatant. Analysis of the secretome of the yeast Pichia pastoris revealed that the most abundant secretory protein, which we named Epx1, belongs to the cysteine-rich secretory protein family CRISP. Surprisingly, the Epx1 secretion leader undergoes a three-step processing on its way to the cell exterior instead of the usual two-step processing. The Kex2 cleavage site within the P. pastoris Epx1 leader is not conserved in the homologues of most other yeasts. We studied the effect of exchanging the Kex2-cleavage motif on the secretory behaviour of reporter proteins fused to variants of the Epx1 leader sequence, and observed mistargeting for some but not all of the variants using fluorescence microscopy. By targeting several recombinant human proteins for secretion, we revealed that a short variant of the leader sequence, as well as the Epx1 signal sequence alone, resulted in the correct N-termini of the secreted proteins. Both leader variants proved to be very efficient, even exceeding the secretion levels obtained with commonly used secretion leaders. Taken together, the novel Epx1 secretion leader sequences are a valuable tool for recombinant protein production as well as basic research of intracellular transport.

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“…GFP is not a natural secretory protein, and it is possible that a small proportion of molecules that fail to fold properly in the environment of the ER are delivered to the vacuole by some form of quality control. Recently, it has been shown that GFP, when fused to the signal peptide of tobacco chitinase, is completely targeted to the vacuole in yeast (Kunze et al 1999). The situation in our case is clearly different, as the majority of mGFP5 is secreted.…”

Section: Discussionmentioning

confidence: 51%

The C-terminal tetrapeptide of phaseolin is sufficient to target green fluorescent protein to the vacuole

Frigerio

Foresti

Felipe

et al. 2001

Journal of Plant Physiology

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SummaryPhaseolin is a vacuolar storage glycoprotein synthesized as a precursor with a short C-terminal propeptide. We have recently shown that deletion of the last four C-terminal amino acids (AFVY, which are part of, or constitute the propeptide) abolishes vacuolar targeting, causing phaseolin to be secreted. Here we provide biochemical and microscopical evidence that the AFVY tetrapeptide, when fused to a secreted version of green fluorescent protein (GFP), inhibits GFP secretion and leads to its accumulation in vacuoles, where it is processed. This demonstrates that the tetrapeptide contains sufficient information for vacuolar sorting.

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The green fluorescent protein targets secretory proteins to the yeast vacuole

Cited by 53 publications

References 47 publications

Demonstration in Yeast of the Function of BP-80, a Putative Plant Vacuolar Sorting Receptor

Demonstration in Yeast of the Function of BP-80, a Putative Plant Vacuolar Sorting Receptor

Multistep processing of the secretion leader of the extracellular protein Epx1 in Pichia pastoris and implications for protein localization

The C-terminal tetrapeptide of phaseolin is sufficient to target green fluorescent protein to the vacuole

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