Site-directed mutagenesis and expression of PC2 in microinjected Xenopus oocytes.

Shennan, K. I. J.; Seal, AJ; Smeekens, Steven P.; Steiner, D F; Docherty, Kevin

doi:10.1016/s0021-9258(18)54384-2

Cited by 36 publications

(4 citation statements)

References 31 publications

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“…It is noteworthy that the islet type II activity (PC2) was optimal at ~pH 6.0--6.2 and at a Ca 2+ concentration compatible with conditions prevailing in the late Golgi apparatus (17). Our demonstration that permeabilized GH3 cells, which express only the PC2 enzyme, require an acidic pH in the TGN for proSRIF processing are quite consistent with these earlier reports and more recent data showing that purified PC1/PC3 and PC2 exhibit acidic pH optima (4,55,74). Surprisingly, there was little or no proSRIF processing at 20°C either in intact GH3.S18.9 cells or in the permeabilized cell system, even after prolonged incubation.…”

Section: Prosrif Cleavage In Permeabilized Cellssupporting

confidence: 91%

Prohormone processing in the trans-Golgi network: endoproteolytic cleavage of prosomatostatin and formation of nascent secretory vesicles in permeabilized cells

Shields

1993

The Journal of Cell Biology

109

154

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Abstract. Many peptide hormones are synthesized as larger precursors which undergo endoproteolytic cleavage at paired basic residues to generate a bioactive molecule. Morphological evidence from several laboratories has implicated either the TGN or immature secretory granules as the site of prohormone cleavage. To identify the site where prohormone cleavage is initiated, we have used retrovirally infected rat anterior pituitary GH3 cells which express high levels of prosomatostatin (proSRIF) (Stoller, T. J., and D. Shields. J. Cell Biol. 1988. 107:2087-2095. By incubating these cells at 20°C, a temperature that prevents exit from the Golgi apparatus, proSRIF accumulated quantitatively in the TGN and no proteolytic processing was evident; processing resumed upon shifting the cells back to 37°C. After the 20°C block, the cells were mechanically permeabilized and pro-SRIF processing determined. Cleavage of proSRIF to the mature hormone was ~35-50% efficient, required incubation at 37°C and ATP hydrolysis, but was independent of GTP or cytosol. The in vitro ATPdepeftdent proSRIF processing was inhibited by inclusion of chloroquine, a weak base, CCCE a protonophore, or by preincubating the permeabilized cells with low concentrations of N-ethylmaleimide, an inhibitor of vacuolar-type ATP-dependent proton pumps.These data suggest that: (a) proSRIF cleavage is initiated in the TGN, and (b) this reaction requires an acidic pH which is facilitated by a Golgi-associated vacuolar-type ATPase. A characteristic feature of polypeptide hormone-producing cells is their ability to store the mature hormone in dense core secretory granules. To investigate the mechanism of protein sorting to secretory granules, the budding of nascent secretory vesicles from the TGN was determined. No vesicle formation occured at 20°C; in contrast, at 37°C, the budding of secretory vesicles was ~40% efficient and was dependent on ATP, GTP, and cytosolic factors. Vesicle formation was inhibited by GTP'yS suggesting a role for GTP-binding proteins in this process. Vesicle budding was dependent on cytosolic factors that were tightly membrane associated and could be removed only by treating the permeabilized cells with high salt. After high salt treatment, vesicle formation was dependent on added cytosol or the dialyzed salt extract. The formation of nascent secretory vesicles contrasts with prosomatostatin processing which required only ATP for efficient cleavage. Our results demonstrate that prohormone cleavage which is initiated in the TGN, precedes vesicle formation and that processing can be uncoupled from the generation of nascent secretory vesicles. residues (20,29). Endoproteolytic cleavage at specific sets of basic residues results in excision of the peptide which may then undergo additional biochemical modifications such as acetylation, amidation, sulfation, etc., to generate a biologically active hormone. Morphological and biochemical evidence suggests that these modifications occur sequentially in the distal secretory pathway, including the T...

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Section: Prosrif Cleavage In Permeabilized Cellssupporting

confidence: 91%

Prohormone processing in the trans-Golgi network: endoproteolytic cleavage of prosomatostatin and formation of nascent secretory vesicles in permeabilized cells

Shields

1993

The Journal of Cell Biology

109

154

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“…When expressed in Xenopus oocytes, pro-PC2 undergoes cleavage at the sequence KRRR 5 6 to generate a 71-kDa intermediate that is further cleaved at the sequence RKKR 84 to generate the 68-kDa mature enzyme. Processing of the 75-kDa propolypeptide can also occur directly by way of cleavage at RKKR 8 4 (Shennan et al, 1991b;Matthews et al, 1994). A similar pattern of processing was shown to occur in GH4 cells (Benjannet et al, 1992) and in islets of Langerhans (Guest et al, 1992).…”

mentioning

confidence: 67%

“…Site-directed Mutagenesis-The PC2 mutants PC2M3 and PC2M4 have been described previously (Shennan et al, 1991b). In PC2M3 the tetrabasic sequence at position 81-84 (where position 1 is the first amino acid in the propolypeptide) was deleted, and in PC2M4 the active-site aspartate was changed to an asparagine.…”

Section: Chemicals and Reagents-[ 3 5smentioning

confidence: 99%

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Differences in pH Optima and Calcium Requirements for Maturation of the Prohormone Convertases PC2 and PC3 Indicates Different Intracellular Locations for These Events

Shennan

Taylor

Jermany

et al. 1995

Journal of Biological Chemistry

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PC2 and PC3, which is also known as PC1, are subtilisin-like proteases that are involved in the intracellular processing of prohormones and proneuropeptides. Both enzymes are synthesized as propolypeptides that undergo proteolytic maturation within the secretory pathway. An in vitro translation/translocation system from Xenopus egg extracts was used to investigate mechanisms in the maturation of pro-PC3 and pro-PC2. Pro-PC3 underwent rapid (t1/2 < 10 min) processing of the 88-kDa propolypeptide at the sequence RSKR83 to generate the 80-kDa active form of the enzyme. This processing was blocked when the active site aspartate was changed to asparagine, suggesting that an autocatalytic mechanism was involved. In this system, processing of pro-PC3 was optimal between pH 7.0 and 8.0 and was not dependent on additional calcium. These results are consistent with pro-PC3 maturation occurring at an early stage in the secretory pathway, possibly within the endoplasmic reticulum, where the pH would be close to neutral and the calcium concentration less than that observed in later compartments. Processing of pro-PC2 in the Xenopus egg extract was much slower than that of pro-PC3 (t1/2 = 8 h). It exhibited a pH optimum of 5.5-6.0 and was dependent on calcium (K0.5 = 2-4 mM). The enzymatic properties of pro-PC2 processing were similar to that of the mature enzyme. Further studies using mutant pro-PC2 constructs suggested that cleavage of pro-PC2 was catalyzed by the mature 68-kDa PC2 molecule. The results were consistent with pro-PC2 maturation occurring within a late compartment of the secretory pathway that contains a high calcium concentration and low pH.

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Biosynthesis of Insulin

Steiner

Chan

Rubenstein

2001

Comprehensive Physiology

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The sections in this article are: Insulin: Properties and Structure Biosynthesis of Insulin Structure and Functions of Precursor Forms Cell Biology Mechanism of Proteolytic Conversion of Proinsulin to Insulin Insulin Storage Vesicles C Peptide, a Co‐secretory Product of the β Cell Regulation of Insulin Biosynthesis The Insulin Gene and its Defects Mutations in the Insulin Gene Defects in Insulin Biosynthesis Prohormone Convertase Defects Conclusion

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Site-directed mutagenesis and expression of PC2 in microinjected Xenopus oocytes.

Cited by 36 publications

References 31 publications

Prohormone processing in the trans-Golgi network: endoproteolytic cleavage of prosomatostatin and formation of nascent secretory vesicles in permeabilized cells

Prohormone processing in the trans-Golgi network: endoproteolytic cleavage of prosomatostatin and formation of nascent secretory vesicles in permeabilized cells

Differences in pH Optima and Calcium Requirements for Maturation of the Prohormone Convertases PC2 and PC3 Indicates Different Intracellular Locations for These Events

Biosynthesis of Insulin

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