Relationship between Cottonseed Malate Synthase Aggregation Behavior and Suborganellar Location in Glyoxysomes and Endoplasmic Reticulum

Abstract

Malate synthase (EC 4.1.3.2) (MS), an enzyme unique to the glyoxylate cycle, was studied in cotyledons of dark-grown cotton (Gossypium hirsutum, L.) seedlings. MS has generally been regarded as a peripheral membrane protein in glyoxysomes and believed by some to be synthesized on rough ER. Immunocytochemical localization of MS in both in situ and isolated cottonseed glyoxysomes, however, showed that MS was located throughout the matrix of glyoxysomes, not specifically associated with their membranes. Biochemical data also supported matrix localization. Isolated glyoxysomes were diluted in variously-buffered salt solutions (200 millimolar KCI or 100 millimolar K-phosphate) or detergents (0.1% Triton X-100, 10 millimolar deoxycholate, or 1.0% Triton X-114) and centrifuged to pellet membranes. Greater than 70% of the MS was recovered in supematants after treatment with salt solutions, whereas generally less than 30% was released following detergent treatments. MS in pellets derived from glyoxysomes burst in low ionic strength buffer solutions was aggregated (observed on rate-zonal gradients). MS released following salt treatments was the 20S nonaggregated form indicating that salt solutions either disaggregated (or prevented aggregation of) glyoxysomal MS rather than releasing it from membranes. We confirmed reports by others that MS comigrated with ER (NADH: cytochrome c reductase) in sucrose (20-40% w/w) gradients buffered with 100 millimolar Tricine (pH 7.5) after 3 hours centrifugation. However, cottonseed MS did not comigrate with ER in gradients buffered with 10 millimolar Hepes (pH 7.0) or 20 millimolar K-phosphate (pH 7.2) after 3 hours centrifugation, or after 22 hours centrifugation in Tricine or Hepes. Collectively, our data with cotton seeds indicate that MS is not a peripheral membrane protein, and that the aggregation behavior of MS (in various buffers) very likely has led to misinterpretations of its putative associations with ER and glyoxysomal membranes.

Characterization of intermediates in the process of plant peroxisomal protein import

“…To investigate the nature of the interactions holding the protein in the membrane, glyoxysomes were allowed to import the fusion protein, lysed as before, then the membranes were re‐isolated and washed with a range of concentrations of sodium chloride or with alkaline sodium carbonate (Figure 5). As controls, malate synthase, a matrix protein peripherally associated with the glyoxysomal membrane membrane (Chapman et al ., 1989), and a 39 kDa integral peroxisomal membrane protein (PMP) (Corpas et al ., 1994) were detected by immunoblotting. Most of the malate synthase is removed by washing the membranes with 0.2 M NaCl and the remainder is removed completely at 0.5 M. Treatment with alkaline sodium carbonate is also effective at completely removing malate synthase from the membrane fraction.…”

Intracellular Localization of Phosphatidylcholine and Phosphatidylethanolamine Synthesis in Cotyledons of Cotton Seedlings

“…CAT activity was high in the soluble region of the gradient as predicted from results of differential centrifugation experiments. The peak CAT activity in approximately 50% sucrose corresponded to intact cottonseed glyoxysomes (5). Two peaks of CCR activity, a major one at about 24% sucrose (ER) and a minor one at about 41% (Fig.…”

“…Some of these data were presented in an earlier report (3). Sucrose Density Gradient Centrifugation Cellular fractions were layered onto 100 mm K-phosphatebuffered (pH 7.2) linear sucrose gradients (33 mL of 20-59% underlaid with 5 mL 59% sucrose) and centrifuged 45 min at 50,000g (4°C) in a Beckman VTi 50 rotor as previously described (5). Fractions (2 mL) were collected using an ISCO model 640 Density Gradient Fraction Collector and analyzed for enzyme activity and protein content.…”

Targeting of glyoxysomal proteins to peroxisomes in leaves and roots of a higher plant.

“…We also note that the MS that cofractionated with mitochondria (fraction 12) was digested with proteinase K, indicating that the protein was associated with but not transported into the organelle. Although MS is a soluble, matrix enzyme, it often aggregates during extraction, and the enzyme aggregates migrate aberrantly in gradients (Chapman et al, 1989).…”