Date of Award

1992

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

First Advisor

Sue G. Bartlett

Abstract

Two protein factors involved in the transport of precursor proteins into intact chloroplasts have been examined. The first of these was observed in experiments utilizing fusion proteins containing various lengths of wheat pS fused to CaM. Transport experiments with these fusion proteins demonstrated a requirement for transport of a pS/CaM fusion protein of a portion of mature S greater than 4 amino acids. Additionally, when the CaM portion of a fusion protein adopted a stable conformation by binding Ca$\sp{2+}$, a larger portion of mature S was needed for efficient transport. A second protein factor was examined using DIDS, and the related compounds H$\sb2$-DIDS, and SITS. Modification of intact pea chloroplasts with these reagents inhibited chloroplast protein transport. This inhibition was not due to disruption of the chloroplast envelope membrane or a non-specific effect on chloroplast metabolism. The level of DIDS required to inhibit chloroplast protein transport correlates well with the level of DIDS which mediates inhibition of chloroplast ATP transport. While DIDS also inhibited chloroplast P$\sb{\rm i}$ transport, the level needed was lower than that required for inhibition of chloroplast protein transport. In addition, the presence of 3-PGA during DIDS modification did not prevent DIDS-based inhibition chloroplast protein transport. The apparent presence of ATP inside the chloroplast and addition of ATP outside the organelle did not relieve inhibition of chloroplast protein transport caused by DIDS. These results indicate that the physical movement of ATP across the chloroplast envelope membrane may be required for chloroplast protein transport. Alternatively, a chloroplast envelope protein may function in a dual capacity to transport ATP and chloroplast precursor proteins. Chloroplast envelope proteins labeled by ($\sp3$H$\sb2$) -DIDS treatment of intact pea chloroplasts demonstrated that the chloroplast phosphate translocator is the most prominently labeled chloroplast envelope protein. However, an additional labeled species was resolved by SDS-PAGE on 12-18% gradient gels containing 7 M urea. This additional labeled protein did not react with antibodies against the spinach chloroplast envelope protein E30 which appear to recognize the pea chloroplast phosphate translocator. This additional ($\sp3$H$\sb2$) -DIDS-labeled protein may be involved in the transport of chloroplast precursor proteins.

Pages

205

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