![]() The cellular organization of various cytoplasmic organelles such as the Golgi and ER seems to be generated and maintained by microtubules (for reviews see Heuser et al., 1989 Kelly, 1990 Cole and Lippincott-Schwartz, 1995). It is reasonable to assume that cytoskeletal elements, in particular microtubules, are a major determinant in these processes. ![]() Not much is known yet about the nature of the factor(s) that determine and sustain peroxisome morphogenesis and their characteristic subcellular distribution. However, a more complex peroxisomal architecture has been encountered when the peroxisomal compartment is going through a phase of rapid growth or is actively engaged in the synthesis of special kinds of lipids ( Yamamoto and Fahimi, 1987 Gorgas, 1987). In mammalian cells, peroxisomes usually appear as single membrane-bound spherical vesicles evenly dispersed throughout the cytoplasm. Peroxisomal membrane proteins are targeted to the organelle via the use of different targeting signals termed membrane PTSs (mPTSs), which have been defined in two proteins ( McNew and Goodman 1996 Wiemer et al., 1996). A second sequence R/K-L/I/V-X 5-H/Q-L/A, called PTS2, is used by a smaller subset of proteins and is found at the NH 2-terminal end of mammalian ( Osumi et al., 1991 Swinkels et al., 1991) and yeast ( Erdmann, 1994 Glover et al., 1994) 3-oxoacyl-CoA thiolase, watermelon malate dehydrogenase ( Gietl, 1990), and amine oxidase of Hansenula polymorpha ( Faber et al., 1994). Earlier work in mammalian cells identified a carboxy-terminal, tripeptide sequence (S/A/C-K/R/ H-L/M), called PTS1, as the major targeting signal used for the sorting of proteins into the peroxisomal matrix ( Gould et al., 1989 Keller et al., 1991). Whether these import pathways are completely distinct or converge to use the same membrane translocation machinery is unknown at present. Genetic and biochemical evidence in yeast and humans supports the notion that there are at least two pathways for the import of proteins into the peroxisomal matrix, each dependent on the use of a specific peroxisomal targeting signal (PTS) 1 and a cognate receptor ( Subramani, 1993). Proteins resident in the peroxisomal membrane and matrix are encoded by nuclear genes and imported posttranslationally ( Lazarow and Fujiki, 1985). ![]() The GFP–PTS1–labeled peroxisomes were found to distribute themselves in a stochastic, rather than ordered, manner to daughter cells at the time of mitosis. High resolution confocal analysis of cells expressing GFP–PTS1 and stained with anti-tubulin antibodies revealed that many peroxisomes were associated with microtubules. In contrast, the microtubule-stabilizing compound paclitaxel, or the microfilament-destabilizing drugs cytochalasin B or D, did not exert these effects. Treatment of cells, transiently expressing GFP–PTS1, with microtubule-destabilizing agents such as nocodazole, vinblastine, and demecolcine clearly altered peroxisome morphology and subcellular distribution and blocked the directional movement. Only the directional type of motion appeared to be energy dependent, whereas the vibrational movement continued even after the cells were depleted of energy. In the latter instance, peak velocities up to 0.75 μm/s and sustained directional velocities up to 0.45 μm/s over 11.5 μm were recorded. Two types of movement could be distinguished: a relatively slow, random, vibration-like movement displayed by the majority (∼95%) of the peroxisomes, and a saltatory, fast directional movement displayed by a small subset (∼5%) of the peroxisomes. The organelle dynamics were examined and analyzed using time-lapse confocal laser scanning microscopy. Peroxisomes in living CV1 cells were visualized by targeting the green fluorescent protein (GFP) to this subcellular compartment through the addition of a COOH-terminal peroxisomal targeting signal 1 (GFP–PTS1).
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |