Mutants blocked in the light reactions of photosynthesis characteristically lack functionally related subsets of thylakoid membrane polypeptides. However, the primary cause and/or mechanism underlying these "losses" is understood only in those cases where chloroplast DNA is known to be missing or altered, or where the synthesis/assembly of chloroplast ribosomes is affected. These losses are especially intriguing in cases where well characterized nuclear mutations lead to the loss of chloroplast-encoded thylakoid polypeptides.

To gain insight into the mechanisms underlying these losses we have compared the synthesis and insertion of chloroplast-encoded polypeptides into wild type thylakoids with thylakoids from the photosystem II deficient hcf*-3 mutant. Hcf*-3 is located on chromosome 1S (MNL 53:37-38); mutation at this locus results in the loss of a prominent 48kD photosystem II reaction center polypeptide (previous article) and a rapidly synthesized 34-32kD, photosystem II associated atrazine binding protein (Leto et al., Plant Physiol. 69:1450-1458; MNL 54:115-117). Both of these polypeptides are chloroplast-encoded and are translated on chloroplast ribosomes.

We conclude from our studies that the apparent "loss" of the 34-32kD and the 48kD polypeptides from hcf*-3 thylakoids is due to accelerated turnover of these polypeptides in the thylakoid membrane. The supporting evidence is as follows:

1) Dot-blot Northern hybridization of total chloroplast RNA against a clone (pZmc427) containing the structural sequence for the 34-32kD polypeptide indicates that hcf*-3 chloroplasts contain wild type amounts of message for this polypeptide.

2) Total chloroplast RNA from both wild type and hcf*-3 seedlings synthesizes a prominent 34kD polypeptide in the rabbit reticulocyte in vitro translation system. It is known that message for the authentic 34-32kD polypeptide is the most abundant message in maize mesophyll chloroplasts.

3) Both the 34-32kD and the 48kD polypeptides are synthesized and inserted into thylakoids when intact hcf*-3 chloroplasts are incubated with 35S-methionine in vitro. Thus, in the absence of the nucleus and over a short (30 min) time course, synthesis and insertion of these polypeptides is essentially normal in hcf*-3.

4) The 34-32kD and 48kD polypeptides are also synthesized and inserted normally into hcf*-3 thylakoids in vivo over the first 1-4 hours that seedlings are incubated with 35S-methionine. However, subsequent "chase" (i.e., dilution) of label in the light reveals a progressive and specific loss of newly synthesized 34-32kD and 48kD polypeptides during the next 20 hours. These losses are also seen when hcf*-3 seedlings are labeled for 1 hour in the light and subsequently transferred to the dark for 20 hours, indicating that photoinhibition is not the primary cause of this accelerated turnover.

5) Turnover of both polypeptides probably occurs in the unpaired (stroma) lamellae, as fractionation studies indicate that neither the 34-32kD or the 48kD polypeptide accumulate in the grana of hcf*-3 thylakoids.

6) The authentic 34-32kD polypeptide lacks lysine residues (McIntosh, J. Cell Biochem. 7B, Abst. 1287). Comparison of in vivo synthesis with 35S-methionine and 14C-lysine indicates that the 34-32kD polypeptide we are following in this study is the authentic chloroplast-encoded species.

While we have apparently discovered the mechanism underlying the "loss" of these polypeptides from hcf*-3 thylakoids, we have yet to discover the primary cause for this turnover. Candidates include increased activity of membrane bound proteases known to specifically metabolize the 34-32kD polypeptide, the involvement of (uncharacterized) nuclear proteins needed for stabilization, or defects in the process of protein import across the chloroplast envelope. We have not yet determined whether the loss of lamellar polypeptides from other hcf mutants is also due to a specific increase in protein turnover.

Kenneth J. Leto, Roslyn Young, Erin Bell* and Lee McIntosh*

*MSU/DOE Plant Research Laboratory, Michigan State

University, East Lansing
 
 

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