A regulator of secretory vesicle size, Kelch-like protein 12, facilitates the secretion of apolipoprotein B100 and very-low-density lipoproteins--brief report

Abstract

Objective: One of the major risk factors for atherosclerosis is the plasma level of low-density lipoprotein (LDL), which is a product of very-low-density lipoprotein (VLDL). Hepatic apolipoprotein B100 (apoB100) is the essential component that provides structural stability to VLDL particles. Newly translated apoB100 is partially lipidated in the endoplasmic reticulum (ER), forming nascent apoB100-VLDL particles. These particles are further modified to form fully mature VLDLs in the Golgi apparatus. Therefore, the transport of nascent VLDL from the ER to the Golgi represents a critical step during VLDL maturation and secretion and in regulating serum LDL cholesterol levels. Our previous studies showed that apoB100 exits the ER in coat complex II vesicles (COPII), but the cohort of related factors that control trafficking is poorly defined.

Approach and results: Expression levels of Kelch-like protein 12 (KLHL12), an adaptor protein known to assist COPII-dependent transport of procollagen, were manipulated by using a KLHL12-specific small interfering RNA and a KLHL12 expression plasmid in the rat hepatoma cell line, McArdle RH7777. KLHL12 knockdown decreased the secreted and intracellular pools of apoB100, an effect that was attenuated in the presence of an autophagy inhibitor. KLHL12 knockdown also significantly reduced secretion of the most lipidated apoB100-VLDL species and led to the accumulation of apoB100 in the ER. Consistent with these data, KLHL12 overexpression increased apoB100 recovery and apoB100-VLDL secretion. Images obtained from confocal microscopy revealed colocalization of apoB100 and KLHL12, further supporting a direct link between KLHL12 function and VLDL trafficking from the ER.

Conclusions: KLHL12 plays a critical role in facilitating the ER exit and secretion of apoB100-VLDL particles, suggesting that KLHL12 modulation would influence plasma lipid levels.

Keywords: COP-coated vesicles; KLHL12; apolipoproteins B; lipoproteins, VLDL.

Figure 1. Effects of KLHL12 knock-down and overexpression on apoB100 and apo100-lipoproteins in McArdle-RH7777 (McA) cells incubated with oleic acid

McA cells were transfected with control or KLHL12-specific siRNA. 48 h later, the cells were incubated with 0.6 mM OA/BSA complex for 1 h, and the incubation was continued for an additional 3 h with ³⁵S-met/cys added to radiolabel apoB100 to steady state. Secreted (A) and intracellular (B) recoveries of radiolabeled apoB100 in the conditioned media and cell lysates were normalized against total TCA-precipitated counts and total protein amounts. (C) Density gradient analysis of secreted apoB100-associated lipoproteins. (D) McA cells were treated as in A and B. Cell homogenates were separated by sucrose density gradient centrifugation to obtain ER microsomes and Golgi membranes, from which apoB100 was immunoprecipitated and quantified. The ER:Golgi ratio of apoB100 recovery was calculated and is displayed. E and F: McA cells were transfected with either Flag-tagged pcDNA3.1-KLHL12 or empty pcDNA3.1 vector (as a control) for 48 h. Cells were then incubated with 0.6 mM OA/BSA complex for 1 h, and the incubation was continued for an additional 3 h with ³⁵S-met/cys to radiolabel apoB100 to steady state. Secreted (D) and intracellular (E) recoveries of radiolabeled apoB100 in the conditioned media and cell lysates were normalized against total TCA-precipitated counts and total protein amounts. A,B,D,E,F: shown are the means, +/− SEM, n=3. *p < 0.05, and **p < 0.01; C: shown are the results representative of 3 independent experiments.

Figure 2. KLHL12 regulates apoB100 metabolism when lipids are primarily endogenously synthesized and co-localizes with apoB100 in the secretory pathway

McA cells were transfected with control or KLHL12-specific siRNA without OA/BSA complexes. 48 h later, cells were incubated with ³⁵S-met/cys for 3 h to radiolabel apoB100 to steady state. Secreted (A) and intracellular (B) levels of radiolabeled apoB100 were decreased similar to that observed in Fig. 1. (C) McA cells were radio-labeled to steady state, and the incubation with the autophagy inhibitor 3-MA increased intracellular apoB100 recovery in KLHL12 siRNA-treated cells. (D) Confocal indirect immunofluorescence images from McA cells probed with antibodies to apoB100 (red), COPII (red), or albumin (red), or KLHL12 (green) as indicated. Arrows in the insets indicate co-localization (yellow) of apoB100, COPII, or albumin with KLHL12. Nuclei were stained with DAPI. Magnification is 63X. The inset is a ×10 zoom of the area of interest. (E) Quantification of KLHL12 co-localization with either albumin or apoB100. A,B,C: shown are the means, +/− SEM, n=3. *p < 0.05, and **p < 0.01. D: shown are the results representative of 3 independent experiments. E: same as A,B,C, but n > 10 cells analyzed for each protein.