Author

Jinzhi Wang

Date

May 2013

Document Type

Dissertation

Degree Name

Ph.D.

Department

Dept. of Cell and Developmental Biology

Institution

Oregon Health & Science University

Abstract

HIGHLIGHTS Two distinct regions (TM1 and cytoplasmic N-terminal domain) in SAC1 (Suppressor of actin mutations 1) control its Golgi distribution A potential oligomerization motif resides within the N-terminal cytoplasmic region adjacent to SAC1-TM1 SAC1, even the Golgi targeted SAC1-K2A, may localize at the cis/medial/trans Golgi, absent from the trans-Golgi network (TGN) 14-3-3 proteins are novel SAC1 interacting partners and may regulate ER exit of human SAC1 SUMMARY At steady state, human lipid phosphatase SAC1 continuously cycles between the ER and cisternal Golgi compartments, which is crucial for the spatial regulation of phosphatidylinositol-4-phosphate (PI4P) at both organelles. In proliferating mammalian cells, a canonical di-lysine motif (583-KEKID-587) at the C-terminus of Sac1 is required for coatomer complex-I (COP-I)-binding and continuous retrieval to the ER. A COP-I binding deficient mutant SAC1-K2A, in which lysines are substituted by alanines, accumulates at the Golgi. However, how the distribution of Sac1 within the Golgi is controlled remains uncertain and the mechanism responsible for Golgi retention of this mutant is unknown. Currently, whether SAC1-K2A mutant localizes at the TGN seems controversial. And it was unclear how ER exit and anterograde transport of SAC1 is regulated. Here, by utilizing the Golgi-targeted SAC1-K2A mutant as a model, we investigated the Golgi retention signal of the human SAC1. We showed that the first of the two transmembrane regions in human SAC1 (TM1) is sufficient for Golgi localization. In addition, we determined that the N-terminal cytoplasmic domain of SAC1 also promotes Golgi localization, even when TM1 is mutated. We conclude that the distribution of SAC1 within the Golgi is controlled via both passive TM1 length-dependent partitioning and a retention mechanism that requires the N-terminal cytoplasmic region. We also identified a potential oligomerization motif within the region adjacent to SAC1-TM1, independent of the N-terminal leucine zipper motif. We found that BFA treatment causes the SAC1-K2A mutant to redistribute into the ER, implying that even the Golgi targeted SAC1-K2A mutant may localize at the cis/medial/trans Golgi instead of the TGN at steady state. Moreover, we found that 14-3-3 proteins are novel SAC1 interacting partners. We showed that binding of 14-3-3 to SAC1 through recognizing a classic mode II-like 14-3-3-binding motif (145-RLSNTSP-151) in the cytoplasmic N-terminal domain of human SAC1. A deletion mutant of SAC1, SAC1(Δ145-151), which lacks the 14-3-3-binding motif, no longer co-immunoprecipitates with 14-3-3 proteins. When over-expressed, all 14-3-3 isoforms, with the exception of 14-3-3ζ, can co-immunoprecipitate with SAC1. A SAC1-S147A/T149A/S150A mutant shows much reduced interaction with 14-3-3, indicating that phosphorylation may be involved in regulating the interaction between SAC1 and 14-3-3. The GFP-SAC1 (Δ145-151) mutant shows a greatly compromised ability to traffic out of the ER compared to wild-type GFP-SAC1. This finding suggests that binding of 14-3-3 may regulate the ER to Golgi transport of lipid phosphatase SAC1.

Identifier

doi:10.6083/M4GM85BZ

School

School of Medicine

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