A Novel Location for Dipeptidyl Aminopeptidase Processing Sites in the Alkaline Extracellular Protease of Yarrowia lipolytica

Matoba, S; Ogrydziak, David M.

doi:10.1016/s0021-9258(18)83309-9

Cited by 33 publications

(6 citation statements)

References 33 publications

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“…We used a pulse-chain regimen followed by immnnoprecipitation to sensitively monitor the biogenesis of AEP in wild-type and secl4rL:: URA3 rL strains. The rate of conversion of pAEP to mAEP, indicative of transit of pAEP from the ER to a late Golgi compartment (Matoba and Ogrydziak, 1989), was quite rapid in wild-type cells (Fig. 8 C).…”

Section: See14 ~ M U T a N T S Exhibit ~L/ild-type Secretory Pathway Functionmentioning

confidence: 95%

“…The rationale for using these primary KEX2p antibodies was that the Y lipolytica XPR6 gene product exhibits several properties that identify it as a KEX2p homolog. First, the XPR6 gene of Y lipolytica encodes an endoprotease that has been implicated in KEX2plike proteolytic processing, at dibasic residues, of the alkaline extracellular protease precursor during its transit through a late Golgi compartment (Matoba et ai., 1988;Matoba and Ogrydziak, 1989). Second, the XPR6p primary sequence is inferred to share significant homology with that of KEX2p (Ogrydziak, D., personal communication).…”

Section: Subceuular Localization Of Sec14p Rtmentioning

confidence: 99%

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A phosphatidylinositol/phosphatidylcholine transfer protein is required for differentiation of the dimorphic yeast Yarrowia lipolytica from the yeast to the mycelial form.

López

Nicaud

Skinner

et al. 1994

The Journal of Cell Biology

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Abstract. The SEC14 sc gene encodes the phosphatidylinositol/phosphatidylcholine transfer protein (PI/PC-TP) of Saccharomyces cerevisiae. The SEC14 sc gene product (SEC14p sc) is associated with the Golgi complex as a peripheral membrane protein and plays an essential role in stimulating Golgi secretory function. We report the characterization of SEC14 ~z, the structural gene for the PI/PC-TP of the dimorphic yeast Yarrowia lipolytica. SEC14 rL encodes a primary translation product (SEC14p vL) that is predicted to be a 497-residue polypeptide of which the amino-terminal 300 residues are highly homologous to the entire SEC14p sc, and the carboxy-terminal 197 residues define a dispensible domain that is not homologous to any known protein. In a manner analogous to the case for SEC14p sc, SEC14p vL localizes to punctate cytoplasmic structures in Y. lipolytica that likely represent Golgi bodies. However, SEC14p vL is neither required for the viability of Y. lipolytica nor is it required for secretory pathway function in this organism. This nonessentiality of SEC14p ~ for growth and secretion is probably not the consequence of a second PI/PC-TP activity in Y lipolytica as cell-free lysates prepared from Asec14 ~z strains are devoid of measurable PI/PC-TP activity in vitro. Phenotypic analyses demonstrate that SEC14p vL dysfunction results in the inability of Y lipolytica to undergo the characteristic dimorphic transition from the yeast to the mycelial form that typifies this species. Rather, Asec14 r~ mutants form aberrant pseudomycelial structures as cells enter stationary growth phase. The collective data indicate a role for SEC14p vL in promoting the differentiation of Y lipolytica cells from yeast to mycelia, and demonstrate that PI/PC-TP function is utilized in diverse ways by different organisms./~ eukaryotic cells have the ability to execute both protein and lipid sorting events. While much has recently been learned about the mechanisms by which proteins traffic between intracellular compartments, or by which proteins are retained in specific organelles, considerably less is known about the intracellular trafficking of lipids. Yet, it is obvious that lipid traffic must also encompass a set of essential cellular activities. For example, intracellular organdies exhibit unique lipid compositions (van Meer, 1989;Pagano, 1990). Moreover, whereas the ER represents the major comparlment of lipid synthesis in the eukaryotic cell, other intracellular comparUnents that experience a

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Section: See14 ~ M U T a N T S Exhibit ~L/ild-type Secretory Pathway Functionmentioning

confidence: 95%

Section: Subceuular Localization Of Sec14p Rtmentioning

confidence: 99%

A phosphatidylinositol/phosphatidylcholine transfer protein is required for differentiation of the dimorphic yeast Yarrowia lipolytica from the yeast to the mycelial form.

López

Nicaud

Skinner

et al. 1994

The Journal of Cell Biology

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“…The digestion was incubated at 37°C over night, an additional 5 WI of dilute endo H was added, and the incubation was continued at 37°C for another 6 h . To terminate the reaction, Laemmli loading buffer (4x) (39) was added, and the mixture was boiled for 5 min .…”

Section: Endoglycosidase H Digestionmentioning

confidence: 99%

“…Y. lipolytica secretes significant levels of several hydrolytic enzymes, and it produces an alkaline extracellular protease (AEP) at levels of 1 to 2 % of total cell protein (40,43) . AEP processing involves several intracellular precursors; the largest and earliest precursor detected in pulse-chase immunoprecipitation experiments is a 55-kD translocated polypeptide which lacks the signal peptide (21, unpublished data) but contains 2 kD of N-linked carbohydrate (39,40) . The next largest AEP precursor is a 52-kD polypeptide which results from dipeptidyl aminopeptidase processing (39).…”

mentioning

confidence: 99%

“…AEP processing involves several intracellular precursors; the largest and earliest precursor detected in pulse-chase immunoprecipitation experiments is a 55-kD translocated polypeptide which lacks the signal peptide (21, unpublished data) but contains 2 kD of N-linked carbohydrate (39,40) . The next largest AEP precursor is a 52-kD polypeptide which results from dipeptidyl aminopeptidase processing (39). The major processing pathway is from the 55-to the 52-kD precursor and then to the 32-kD mature AEP (40).…”

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A mutation in the signal recognition particle 7S RNA of the yeast Yarrowia lipolytica preferentially affects synthesis of the alkaline extracellular protease: in vivo evidence for translational arrest.

Yaver

Matoba

Ogrydziak

1992

The Journal of Cell Biology

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Abstract. Replacement of the signal recognition particle (SRP) 7S gene (SCRI) on a replicating plasmid with scrll (G to A at 129 and A to T at 131 in the consensus sequence -GNAR-in the loop of domain III) resulted in temperature sensitivity for growth of cells in which both chromosomal SRP 7S RNA genes were deleted . Pulse-chase immunoprecipitation experiments were done after a shift to non-permissive temperature using the major secreted protein the alkaline extracellular protease (AEP) as a reporter molecule. No untranslocated AEP precursor was detected in a strain with scrll on a plasmid, but the amount of the largest AEP precursor (55 kD) immunoprecipitated as a percentage of total protein synthesized was reduced 68% compared to an isogenic strain with SCRI N higher eukaryotes, evidence suggests that the signal recognition particle (SRP)' is essential for protein translocation across the ER membrane. SRP is a soluble IIS ribonucleoprotein composed of six polypeptides (72,68,54,19,14, and 9 kD) and a single 7S RNA of 300 nucleotides (66,74) . From the study of in vitro systems, a model has been proposed in which SRP functions as an adapter between the translational machinery in the cytoplasm and the translocational machinery in the ER membrane (49,66,74) . The functions of individual SRP proteins have been elucidated by the study of "mutant" SRPs reconstituted in vitro (63, 65) . Three functions ofSRP have been identified : signal recognition, elongation arrest, and translocation promotion (66). Translation of mRNAs coding for secretory proteins begins on free ribosomes in the cytoplasm . When the polypeptide chain has elongated sufficiently, SRP binds the signal sequence/ribosome complex . This interaction results in arrest or pausing of translation (74, 78) . The SRP/ribosome complex is then targeted to the SRP receptor, an ER integral membrane protein. This results in the release of SRP and translational arrest, and the polypeptide is then co-translationally translocated into the ER. During or shortly after on the plasmid . The possibility that an untranslocated precursor was synthesized but not detected because of instability was largely eliminated by detection of a 53-kD untranslocated precursor of a mutated AEP (P17M; methionine replaced proline in the second position of the pro-peptide) which chased to the 55-kD translocated AEP precursor. Thus, SRP has a role in the biosynthesis of AEP. Possibly, the scrll mutation does not affect signal recognition or translational arrest but instead results in maintenance of translational arrest of AEP synthesis . The results also suggest that AEP can be translocated in vivo either co-translationally in which SRP is at least involved in biosynthesis or posttranslationally without SRP involvement .translocation, signal peptide cleavage and core glycosylation occur. Besides a role as a scaffold for binding SRP proteins (73), functional roles for SRP 7S RNA in elongation arrest and in interaction with the SRP receptor have been proposed (36,63,81) . Recently, it has b...

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Secretion and pH-dependent self-processing of the pro-form of the Yarrowia lipolytica acid extracellular protease

McEwen

Young

1998

Yeast

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A Novel Location for Dipeptidyl Aminopeptidase Processing Sites in the Alkaline Extracellular Protease of Yarrowia lipolytica

Cited by 33 publications

References 33 publications

A phosphatidylinositol/phosphatidylcholine transfer protein is required for differentiation of the dimorphic yeast Yarrowia lipolytica from the yeast to the mycelial form.

A phosphatidylinositol/phosphatidylcholine transfer protein is required for differentiation of the dimorphic yeast Yarrowia lipolytica from the yeast to the mycelial form.

A mutation in the signal recognition particle 7S RNA of the yeast Yarrowia lipolytica preferentially affects synthesis of the alkaline extracellular protease: in vivo evidence for translational arrest.

Secretion and pH-dependent self-processing of the pro-form of the Yarrowia lipolytica acid extracellular protease

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