The general goal of these studies is to identify and characterize proteins and protein complexes that serve as morphogenetic factors in membrane assembly. In particular, we are interested in membrane-associated factors that control the differentiation of chloroplast membranes into appressed (grana) membrane regions, and non-appressed (stroma) membrane regions. Chloroplast membranes are an excellent model system for the investigation of membrane morphogenesis, both because of their characteristic architecture, which is controlled by genetic and environmental factors, and because they are the best studied membrane system from a biochemical, structural and genetic perspective. Proteins associated with the photosystem II-related light harvesting complexes (LHC II's) appear to play a major role in the formation of appressed membrane regions. These are encoded by two large, highly homologous gene families. We have developed an improved native gel system for membrane protein complexes which allows us to separate up to ten LHC II chlorophyll-protein complexes, there by providing the opportunity to correlate the appearance and accumulation of individual complexes with the formation of specific membrane configurations. We will prepare nonspecific antibodies against synthetic peptides corresponding to unique sequence domains of LHC II genes, and use these antibodies to study the composition of different LHC II complexes, the hierarchy of their appearance during development, and their degradation during adaptation to different light environments. We have also identified a set of ch1 b-deficient mutants with altered LHC II patterns exhibiting different structural phenotypes. We propose to associate these mutants with particular blocks in the chlorophyll biosynthesis pathway. A second line of work will focus on the composition and assembly of six different photosystem I-related complexes that we can also separate on our improved native gel system. Preliminary experiments show that this gel system has great potential for analyzing photosystem I-defective mutants in Chlamydomonas.
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