Molecular organization and trafficking of membrane proteins

Membrane proteins comprise about 30% of proteome. Molecular organization and trafficking of membrane receptors and ion channels are important in many biological processes. Potassium (K+) channels, a regulator of the excitability of neurons, are composed of various K+ channel families, reflecting a diversity of specific signaling requirements. My thesis research attempts to address how functional diversity and plasticity of ion channels and membrane receptors can be achieved. Functional diversity suggests multiple levels of regulation including differential subunit assembly and association with interacting proteins. The native polypeptide of a neuronal delayed rectifier, Kv2.1, has not been purified. Therefore, composition of the Kv2.1 channel complexes was not well understood. In Chapter I, I report biochemical characterization of Kv2.1 channel complexes. They behave as large macromolecular complexes with an apparent oligomeric size of 650 kDa by gel filtration. Distinct populations of native Kv2.1 complexes from rat brain were detected, suggestive of biochemical heterogeneity. Immunoaffinity purified Kv2.1 complexes were predominantly composed of Kv2.1 but not homologous Kv2.2 subunits. The interacting proteins and their physiological relevance are of interest for further investigation.; Proteins have distinct biochemistry at their termini. Sequence motifs at carboxyl-termini in linear polypeptides are uniquely positioned and functionally serve as recognition signatures. At the proteome level, it is unknown whether and what C-terminal sequences might be particularly conserved, which may be related to specific biological functions. To investigate this question, terminal sequences of entire proteomes from Saccharomyces cerevisiae and various organisms were analyzed. The results summarized in Chapter II show that there are both known and novel terminal sequences. Their frequency in proteome was similar to that of a well known ER localizing HDEL signal. These findings support the notion that there may be additional C-terminal signals and the conserved motifs could be experimentally tested for currently unknown functions.; For plasma membrane proteins, their density on the cell surface is often a key determinant of their overall functions, hence regulated by various means. Sorting and trafficking by signal motifs is one of the mechanisms. When diversity space of random sequences was tested in a genetic screen for forward trafficking activity, a novel C-terminal SWTY motif was isolated. SWTY is capable of overriding ER localization and directing cell surface expression. Phosphorylation-dependent recruitment of 14-3-3 proteins is required for the potentiation of surface expression. However, the kinase(s) for SWTY sequence has not been identified. In Chapter III, I report kinase-activity dependent regulation of trafficking of membrane proteins. I demonstrate that the inducible surface expression is regulated by protein kinase B (Akt) activity. SWTY-like motifs are found in native receptors including GPR15, a human immunodeficiency virus (HIV) coreceptor. I further show that Akt elevates its surface expression, suggesting a role of Akt pathway in HIV infection. The findings in the study show that functional diversity of ion channels and membrane receptors can be achieved by forming macromolecular complexes with interacting proteins. The sequence selectivity and the coupling of posttranslational modification confer plasticity by signal-induced trafficking.